How Multiplexing Distorts Metabolic Phenotyping Data

Multiplexing metabolic signals causes significant data distortion. This post explains how, and gives you access to an amazing, interactive tool for exploring the metabolic data distortions produced by this practically universally-used technique!

To save cost, almost all metabolic phenotyping systems multiplex their gas analyzers, so that the air streams from the metabolic chambers are analyzed in succession. As a result, it takes an appreciable time for the gas analyzers to sample repeatedly from a given cage. The time between successive samples from a given cage is referred to as the cycle time. For 16 animals, the cycle time can vary from 5 minutes (Promethion-M system at its fastest recommended setting) to over 40 minutes (systems from other manufacturers).

Multiplexing distorts metabolic data in three primary ways.

1. The cycle time imposes a limit to the fastest events that can be recorded. If a metabolic event occurs between two cycles, it will not be detected at all. As a result, multiplexed data always overestimates resting energy expenditure (REE) and underestimates active EE (AEE).
2. If an animal changes its EE in a cyclical fashion, multiplexing can severely distort metabolic data through a phenomenon known as aliasing. This can cause enormous under- or over-estimates, particularly of AEE but potentially of REE as well.
3. The point in time at which the recording starts will significantly affect the measured metabolic data. This effect is purely stochastic and out of the control of the researcher, because it relates to phenomena in the future.

The only way to avoid these problems entirely is to record metabolic data continuously. The only commercially available system capable of this feat for multiple animals is the Promethion-C system Continuous metabolic phenotyping system (brochure here). It has one-second time resolution for metabolic data (and all other types of data) from an essentially unlimited number of animals. It is an up-and-running, fully documented system in active production - not an experimental prototype will-o'-the-wisp that dooms researchers to month after month of painful, time-sucking frustration and wasted effort (believe me, we've heard the stories). Without Promethion's advanced proprietary technologies I seriously do not think that the parallel, continuous approach is feasible. You can read a bit more about multiplexed vs. continuous metabolic measurement systems in this poster (AALAS, 2012).

We have created an interactive tool to allow you to explore the metabolic data distortions caused by multiplexing. Before clicking on the link, please read a bit about how the tool works.

• The page will take a little while to load, because it is accompanied by a substantial data file.
• The interactive graph is optimized for a monitor resolution of 1700 pixels. If you have a smaller monitor you will need to scroll about a bit to see (for example) the Y axis on the right side of the graph.
• To set the start point for the multiplexing, click on the graph. The simulated multiplexing will start at that time on the X axis.
• To change the cycle time of the simulated multiplexed system, either drag and drop a multiplexed data point, or enter a new cycle time manually in the Cycle Time box.
• If the graphic doesn't work for you or simply doesn't appear, ensure that JavaScript is enabled on your browser, and/or you may need to update your browser.

Here is the visualization tool. Enjoy!

For more on the future of metabolic phenotyping, see The Future of Metabolic Phenotyping!

If you have any questions, contact me.

* Thanks to Thomas Förster, Ph.D., Sable Systems International's expert in-house data analysis and data presentation consultant, for creating the JavaScript application.