Background: Besides plants and animals, nearly all of us produce it endogenously. It contains a C₅ hemiterpene unit essential for the biosynthesis of steroid hormones and terpenoids. It is the second most abundant volatile organic compound (VOC) in our breath and we continuously exhale it in trace amounts¹. Thus, isoprene (1,3-pentadiene, C₅H₈) holds potential importance in clinical, pre-clinical, botanical and environmental sciences.

<1% of our breath contains thousands of VOCs in trace concentrations. Since the dawn of modern breathomics, exhaled C₅H₈ has been described as a potential biomarker for diseases such as hypercholesterolemia, metabolic stress, muscle dystrophy, cardio-pulmonary diseases and various forms of cancers. Concentration changes are also related to various physiological and metabolic effects, inherited errors and to our biological ageing process². Despite such attractiveness, until now this VOC could not be used in routine clinical practice as a biomarker. This was mainly due to its uncertain metabolic origin and biological functions involved in humans.  

The putative origin: 39 years ago, Deneris et al hypothesized that cholesterol biosynthesis in the liver potentially gives rise to isoprene in our breath³. The belief was based on pre-clinical in vitro experiments. Nevertheless, changes and/or differences observed in breath isoprene under various conditions investigated in clinical studies could not be justified by the suggested origin.

Though a few studies raised doubts about the hepatic source, concrete downstream proof was unavailable^4–6. While absence of non-sterol isoprene is characterized as an indicator for inherited life-threatening conditions, since 1981, there were random reports of rare encounters of isoprene absent healthy adults. Such adults are rare (<0.3% prevalent) in the nature and I was eager to find one as the key to isoprene’s origin.

Conception: Since 2013, I was investigating the dynamic nature of exhaled VOCs under varying physio-metabolic effects. On a fine morning in May 2015, a young and healthy German lady walked into our labs to participate in an ongoing breath test. As soon as she started breathing into our real-time mass-spectrometer, the exhaled isoprene signal completely vanished. Driven by excitements, I had a meeting with my mentors (Schubert and Miekisch). Along with the colleagues from the Hematology-Oncology Group, we embarked into down-stream investigations.

In 2021, we published the first down-stream analysis on the isoprene absent adult and her blood-relatives that disqualified the putative origin of isoprene⁷. Interestingly, her parents and sibling sister turned out as isoprene deficient – indicating a recessive trait. Nevertheless, our way to its true origin was still far away and a single case was insufficient for multi-omic investigations. So, we kept on searching!

Multi-omic investigations: Meanwhile, in consecutive clinical screening studies, we identified four more healthy German adults without breath isoprene. Therefore, to reveal the human origin, whole exome sequencing was planned to identify unknown homozygous variants shared by these five (amongst 2000 recruitments) rare adults. Shared mutations must be checked via targeted sequencing in blood-related and unrelated (isoprene normal) adults. Plasma lipids, metabolites and hormones related to cholesterol metabolism should be examined serologically.

Alas! we had no direct funding available for this study. To ensure that we can execute all experiments on time, my mentors readily gathered the required funds from other projects and the Hematology-Oncology colleagues provided the necessary wet-lab facility and materials. We were ready!

After having the ethical clearance, we reinvited the rare subjects. We posted official invitations to their letterboxes with the hope that they might respond. After a week of dead silence, all positive responses appeared. We were thrilled and witnessed the scientific motivations of them and their blood-relatives – few even travelled from hundreds of kilometers away. In order to accommodate them flexibly, we conducted all experiments during the weekends. Signed informed consents were obtained prior to participation.    

Findings: Amongst all homozygous (100% allele frequency) mutations with <1% European prevalence, an isopentenyl-diphosphate delta isomerase-2 (IDI2) stop-gain mutation was shared between the rare adults revealing the genetic origin of isoprene absence. This mutation caused losses of enzyme active sites and affected the magnesium ion–cofactor binding sites. IDI2 variant was heterozygous in their isoprene deficient family members and was absent in unrelated adults. No considerable serological aberration or mutation was found in the isoform IDI1 in any participant.

Human IDI2 is highly expressed only within the peroxisome (lipolytic organelle) of skeletal-myocytes, while expression of IDI1 is conserved in various tissues and mainly in the liver. Although, IDI1 may produce some amount of isoprene in the liver, cytochrome-P450 enzymes of human hepatocellular microsomes immediately oxidize isoprene. Preclinical studies have shown that unlike other mammals, pigs and bottle-nose dolphins do not exhale isoprene. We searched and found that these animals do not express IDI2. 

Inferences: Skeletal muscles metabolize lipids (oxidize cholesterol and fatty acids) to produce energy, regulate intramyocellular signaling and integrity. Beta-oxidation in myocellular peroxisomes produces acetyl-CoA i.e., channeled towards dimethylallyl diphosphate [(C₁₄)DMAPP] production. As human IDI2 catalyzes the isomerization of isopentenyl diphosphate [(C₁₄)IPP] to highly nucleophilic DMAPP the origin of human isoprene is related to muscular metabolism rather than the suggested cholesterogenesis in the liver.

The well-known instant rise in breath isoprene during muscle activity (movement/exercise) along with our present findings underpin its actual origin from lipolysis in the skeletal muscles. In plants, only DMAPP is converted to isoprene via isoprene synthase enzyme. As humans do not have isoprene synthase or its enzyme homologues, the wild type IDI2 gene and enzyme turned out as the rate limiting factor for human isoprene production⁸.

Implications: Our discovery of the genetic origin and metabolic routes of human isoprene production enabled objective interpretations and applications of isoprene as a noninvasive biomarker for various conditions of clinical interest and elucidated new frontiers in sports and musculoskeletal medicine.

Perspective: Knowledge of accurate metabolic origin is indispensable for valid clinical interpretation of any endogenous biomarker. Apart from isoprene, our breath contains many potentially endogenous VOCs and their sources are yet uncertain. We cannot speculate on clinical outcomes based on putative substance origins as such practice inevitably is prone to error.

This is just the beginning and our search is on!

References:

1. Mochalski, P., King, J., Mayhew, C. A. & Unterkofler, K. A review on isoprene in human breath. J. Breath Res. (2023) doi:10.1088/1752-7163/acc964.
2. Miekisch, W., Sukul, P. & Schubert, J. K. Chapter 2 Origin and Emission of Volatile Biomarkers in Breath: Basics and Dynamic Aspects. in Volatile Biomarkers for Human Health: From Nature to Artificial Senses 22–38 (The Royal Society of Chemistry, 2023). doi:10.1039/9781839166990-00022.
3. Deneris, E. S., Stein, R. A. & Mead, J. F. Invitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction. Biochem. Biophys. Res. Commun. 123, 691–696 (1984).
4. Turner, C., Spanel, P. & Smith, D. A longitudinal study of breath isoprene in healthy volunteers using selected ion flow tube mass spectrometry (SIFT-MS). Physiol. Meas. 27, 13–22 (2006).
5. Miekisch, W., Schubert, J. K., Vagts, D. A. & Geiger, K. Analysis of volatile disease markers in blood. Clin. Chem. 47, 1053–1060 (2001).
6. King, J. et al. Breath isoprene: Muscle dystrophy patients support the concept of a pool of isoprene in the periphery of the human body. Biochem. Biophys. Res. Commun. 423, 526–530 (2012).
7. Sukul, P., Richter, A., Schubert, J. K. & Miekisch, W. Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin. Heliyon 7, e05922 (2021).
8. Sukul, P., Richter, A., Junghanss, C., Schubert, J. K. & Miekisch, W. Origin of breath isoprene in humans is revealed via multi-omic investigations. Commun. Biol. 6, 1–12 (2023).