Driving research of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME / CFS),
Post Treatment Lyme Disease Syndrome (PTLDS), Fibromyalgia and Long Covid

Nitrogen Metabolism and
Testing Nitrogen Hypothesis in ME/CFS

This project aims to test the nitrogen hypothesis, which is that damaging, nitrogen-containing by-products of energy metabolism accumulate more readily in the cells of ME/CFS patients.

  • Christopher Armstrong, PhD
  • Ronald Davis, PhD
  • Paul Gooley, PhD
  • Neil McGregor, PhD

Of particular interest are the mitochondria and their role in energy production and resolution of the inflammation that is generated by muscular stress and exercise. It is both possible and even likely that clues for potential biomarkers for PEM will be revealed in these highly detailed studies. 


We hypothesize that toxic nitrogen-containing by-products of energy metabolism accumulate more readily in the cells of ME/CFS patients. This accumulation, in turn, creates a cycle of energy generation to overcome a stressed state while generating more toxic nitrogen by-products in the process. We will also identify the compounds that can circumvent the production of toxic nitrogen by-products and release patients from this cycle.


To test this hypothesis, we will culture white blood cells in growth media with added nitrogen-labelled amino acids and measure the nitrogen-labelled by-products of energy metabolism. Cells from 30 ME/CFS patients and 20 age and sex-matched controls will be collected and processed at the Stanford Genome Technology Center and cultured for our nitrogen tracking experiment. These experiments will be conducted on blood cells directly from ME/CFS patients and cells cultured from ME/CFS patients.

To capture whether there is an ammonia accumulation in vivo, our best available option is observing the urine concentration of nitrogen-containing metabolites over a short time-course in which varying levels of physical/mental activity have occurred. The 30 ME/CFS patients and 20 sex-match control subjects from the above experiment will be required to provide all urine samples over 4 consecutive days (~20 samples per individual) while their exertion and heartbeat data is collected using wearable technology. Normally, most nitrogen by-products are converted to primary urea along with amino acids, peptides, creatine, creatinine, and polyamines.

In a pathological state, highly reactive, toxic nitrogen by-products are excreted in the urine as nitrate, nitrite, and ammonia. We will measure the ratios of healthy to pathological nitrogen metabolites in the urine and correlate this with the metadata activity.