The completion of the Human Genome Project and the advent of low-cost genomic sequencer technology changed the direction of biological analysis and metabolomics. Biomarkers have always been a target in disease research, but today’s technologies allow researchers to extract more information than ever before.
While bodily fluids like blood and urine are common samples for biomarker analysis, breathomics—or the testing of breath—has gained rapid research interest in the genomic era. Of course, breathomics is not new. Somewhere in the 5th century BCE, Hippocrates tied “sweet smelling” breath to diabetes, even if he didn’t fully understand the implications of his discovery at the time. Since then, breath testing has been used as a rapid, non-invasive test for everything from disease to drug and alcohol intoxication.
Researchers at the National Institutes of Health (NIH) have now added to the list, designing a breath test to better assess patients with methylmalonic acidemia (MMA), a rare genomic disease that impairs the body’s ability to metabolize certain proteins and fats. This causes toxic substances to build up, resulting in kidney disease, pancreatitis, movement disorders, intellectual impairments, complications and, in severe cases, death.
Currently, MMA is incurable, but patients manage their symptoms through diet and vitamins. However, some individuals need liver or combined liver and kidney transplants to help restore the body’s normal level of metabolic proteins.
The test, which focuses on the MMUT protein, not only measures how well patients with MMA respond to organ transplantation, but it can also be used to assess the severity of the disease. An accurate severity level assessment can help determine if a patient would benefit from surgical or experimental genomic therapies.
“Vast fluctuations in metabolic substances in the bodies of patients make it difficult for us to tell if treatments like genome editing and transplants are likely to be successful,” said Charles Venditti, M.D., senior author and senior investigator at the National Human Genome Research Institute (NHGRI). “Instead of looking at levels, we decided to measure metabolism itself.”
In healthy persons, the MMUT protein helps break down food into a chemical byproduct called propionate, which the human body follows with oxidation. Through oxidation, a person converts propionate into energy and exhaled carbon dioxide. However, MMUT protein function is compromised in people with MMA.
Thus, Venditto and his team turned to breathomics to measure exhaled carbon dioxide as a means to track oxidation of propionate in a non-invasive way. This would act as a proxy for how much oxidation was occurring—or not occuring—in a patient’s body, the researchers explained.
First, however, the researchers had to figure out how to distinguish exhaled carbon dioxide released as a result of propionate breaking down in the body versus exhaled carbon dioxide released from various other metabolic processes.
Typically, humans exhale carbon dioxide that contains a light, common form of carbon—carbon 12. So, to detect if the MMUT protein was functioning properly, researchers focused on detecting a heavier, less abundant version of carbon—carbon 13—which is a commercially available food additive.
For their study, published in Genetics in Medicine, the research team recruited 57 participants, including 19 MMA patients who had received transplants (liver, kidney or both) and 16 healthy volunteers. They gave the volunteers a dose of carbon 13 via a drink or through a feeding tube, then collected breath samples after 2 minutes.
According to the study results, MMA patients who did not receive any treatment had lower levels of carbon 13 than healthy volunteers. Meanwhile, MMA patients with liver transplants had higher levels of carbon 13, similar to the healthy volunteers. This research team says this indicates that the MMUT protein was helping oxidize the carbon 13 molecules by bonding with inhaled oxygen molecules. Additionally, higher levels of carbon 13 oxidation correlated with better clinical outcomes, such as improved cognition and slower decline in kidney function.
“Our next goal is to see if this specialized breath test can detect increase in carbon 13 propionate oxidation after gene, mRNA or genome editing therapies,” Venditti said. “This way, we can also use this test to measure how effective these treatments are in restoring MMUT function.”
Currently, the breath test is only available at the NIH Clinical Center; however, Venditti and his team are interested in making it broadly accessible for both research and clinical use.
Photo: Breath test for methylmalonic acidemia measures disease severity and success of liver transplantation for patients. Credit: Ernesto del Aguila III, NHGRI