Mitochondrial Testing

A vital dimension in diagnosing cellular health

Our mitochondria are key orchestrators of cellular health, responsible for our energy production, biosynthesis of cellular components, and cellular signalling. It has always been very difficult to measure the activity of these infinitely small cell organelles.

The Seahorse XF (please see “Energy pathways” video below) has opened up new diagnostic vistas: high-throughput respirometry has been developed that can dynamically assess the response of mitochondria to changes in their cellular environment. Multiple mitochondrial parameters are available that have the power to predict both disease progression and response to therapy. AONM is now able to offer a range of these new tests.

What tests are available?

Test 1. ATP profile

The ATP Profile determines the total ATP production and allows for a quantitative comparison of the cells’ energy engines – mitochondria and glycolysis. The ATP Profile consists of the following markers:

a) Total ATP
The maximum quantity of ATP that the cells are able to produce at rest via both mitochondrial and non-mitochondrial pathways. Total ATP is all the adenosine triphosphate (our cells’ energy currency) available to the cell. This makes it possible to assess the relative performance of anaerobic glycolysis versus mitochondrial respiration.

b) Mitochondrial ATP (also called ATP-linked respiration)
This is the rate of mitochondrial aTP synthesis in a defined basal state. An increase in the ATP-linked oxygen consumption rate would indicate an increase in ATP demand, whereas a decrease would indicate low ATP demand, lack of substrate availability, and/or damage to oxidative phosphorylation.

c) Glycolytic ATP
ATP can also be produced in the cytosol, outside the mitochondria (though still inside the cell). This is produced solely from glucose, and the amount of ATP per molecule of glucose is very low (just 2 ATP per molecule of glucose). This parameter measures the glycolytic capacity for ATP production: the maximum quantity of ATP that the cells are able to produce at rest via non-mitochondrial pathways, i.e. anaerobic glycolysis. This makes it possible to assess the relative performance of anaerobic glycolysis versus mitochondrial respiration.

d) Reserve capacity
Reserve capacity is the difference between the basal and the maximum respiration rate of the cell. It is a measure of the maximum potential respiration capacity that the cell can utilise under conditions of stress and/or increased energetic demand. Any lack of reserve respiratory capacity generally indicates mitochondrial dysfunction.

Click here to see a sample ATP Profile report

Test 2. Mitochondrial Health Index

The mitochondrial health index (MHI) in the circulating mononuclear cells of the blood (PBMCs) is a dynamic index composed of the parameters listed below. It can be used to measure improvement in mitochondrial function, and to help identify where the block to optimal functioning might lie.
a) Basal respiration rate

Basal respiration can be considered a threshold below which the cell cannot sustain oxidative phosphorylation to meet energy demand. Basal respiration is usually controlled strongly by mitochondrial ATP turnover and partly by proton leak .

b) Mitochondrial ATP turnover

This is the rate of mitochondrial ATP synthesis in a defined basal state. An increase in mitochondrial ATP turnover indicates an increase in ATP demand, e.g. by stimulation of ion pumps or metabolic cycles, whereas a decrease indicates low ATP demand, a lack of substrate availability and/or damage to oxidative phosphorylation.

c) Proton leak

This parameter reflects movement of any cations [click here for further explanation) or substrates across the mitochondrial inner membrane, including protons. An increase in proton leak is consistent with the mitochondria becoming less efficient.

d) Maximum respiration rate

This rate gives the maximum oxygen consumption that can take place at Cytochrome c oxidase (Complex IV), whether limited by availability of substrate or activity of the electron transport chain. A decrease in maximum respiratory capacity is then a strong indicator of  potential mitochondrial dysfunction.

e) Reserve capacity

The difference between the basal respiration and maximum respiration rate is the reserve capacity of the mitochondrion, which is a measure of the maximum potential respiratory capacity the cell can utilize under conditions of stress and/or increased energetic demand. The lack of reserve respiratory capacity indicates a mitochondrial dysfunction that may not be particularly apparent under basal conditions.

f) Non-mitochondrial respiration rate

This parameter is an index of oxygen-consuming processes that are not mitochondrial. In leucocytes, the non-mitochondrial respiration rate is typically attributed to enzymes associated with inflammation, including cyclooxygenases, lipoxygenases and NADPH oxidases. An excessive level is regarded as a negative indicator of mitochondrial health.

g) Calculation of the MHI

This is calculated from the various parameters measured in the test, as follows:

MHI = log (reserve capacity) x (mitochondrial ATP turnover)
                        (non-mitochondrial) x (proton leak)

The figure calculated for the MHI provides a marker against which the impact of therapy can be measured. For further information, please refer to the seminal article by Chacko et al [i] It was on the basis of the work of Chacko et al at the University of Alabama that this metric was developed (see below for further information, it is well substantiated and recognised internationally).

Click here to see a sample Mitochondrial Health Index report

Tests 1 and 2 can be combined for a discounted price.


Supplementary Biomarkers

(Must be ordered at same time as ATP or MHI tests)

Ratio of mtDNA to nDNA

The ratio of mtDNA (mitochondrial DNA) to nDNA (nuclear DNA) makes it possible to calculate the number of mitochondria per cell. The amount of mitochondria decreases physiologically with age. However, various metabolic or neurodegenerative diseases are also associated with reduced numbers of mitochondria, which can be the cause of ATP deficiency.


PGC-1α stands for Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1Alpha. This is a transcriptional coactivator which is of central importance for the induction of mitochondrial biogenesis.


NRF-2, nuclear factor erythroid 2-related factor 2, is a transcription factor whose physiological role is to protect cells from reactive oxygen species.

Lactate/pyruvate ratio

The lactate to pyruvate (L:P) reflects the equilibrium between the product and substrate of the reaction catalyzed by lactase dehydrogenase and indirectly reflects the NADH: NAD+ cytoplasmic redox state. An excess of lactate indicates poor mitochondrial function.


How are the tests conducted?

AONM is working with a laboratory in Magdeburg, Germany, called MMD, Magdeburg Molecular Detections that specialises in mitochondrial testing. MMD is a pioneer in the use of the Seahorse XF. Seahorse Biosciences has developed a unique extracellular flux analyser that is able to measure the two major energy-producing pathways of the cell: mitochondrial respiration and glycolysis. They use a microplate-based system with unprecedented throughput to make these measurements very sensitively, with extremely rapid kinetics. See these videos below for the full explanation:

How the Seahorse XF works

Seahorse: Energy pathways

The tests require only one vial of blood in a CPDA[i] tube. The laboratory uses PBMCs (peripheral blood mononuclear cells, no centrifuging); it is important that they reach Germany quickly. AONM supplies the kit and organises the shipping.


How did the Mitochondrial Health Index evolve?

Researchers at the University of Alabama developed a new set of biomarkers called the Bioenergetic Health Index (BHI) that measures mitochondrial dysfunction, indicating a susceptibility to certain disorders. The sequential addition of well-characterised modulators of oxidative phosphorylation (see more explanation here) allow a mitochondrial profile to be measured in cells (Figure 1). The relation of the different markers to each other reveals crucial information about cellular performance along a sliding scale from optimal to suboptimal. We have termed this the Mitochondrial Health Index rather than “Bioenergetic” Health Index to make it clearer what the main thrust of the parameters is – the mitochondria.

Fig 1. The Bioenergetic Health Index as a dynamic measure of the response of the body to stress

OCR = oxygen consumption rate
Basal = basal respiration;
AL = ATP-linked respiration (mitochondrial ATP turnover)
PL = proton leak; non-mitochondrial = non-mitochondrial oxygen consumption rate
Maximal = maximum oxygen consumption rate
ROS = reactive oxygen species
OxPhos = oxidative phosphorylation
ATP = adenosine triphosphate


Source: Chacko BK, Kramer PA, Ravi S, Benavides GA, Mitchell T, Dranka BP, Ferrick D, Singal AK, Ballinger SW, Bailey SM, Hardy RW, Zhang J, Zhi D, Darley-Usmar VM. The Bioenergetic Health Index: a new concept in mitochondrial translational research. Clin Sci (Lond). 2014 Sep;127(6):367–73

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