For the last 39 years, we have erroneously regarded hepatic cholesterogenesis (producing > 90% of human cholesterol) as the prime origin of human exhaled isoprene. This believe was based on in vitro synthesis of isoprene from DL-mevalonate by utilizing a rat liver cytosolic fraction 30. Consequently, various physio-metabolic and clinical conditions driven interesting differences (cross-sectional) and/or changes (longitudinal) in isoprene exhalation could not be explained via the well-known/established effects of those conditions on hepatic cholesterogenesis. As a result, breath isoprene could not step into routine clinical practice as a noninvasive biomarker. In 2021, we challenged the putative origin of breath isoprene 29 and here, we have discovered the actual origin of human exhaled C5H8 by multi-omic analysis of genes and metabolites.
Distribution of exhaled isoprene concentrations from 2000 screened subjects reconfirmed previously reported age dependency 21–23, 29 of its exhalation. Complete absence of exhaled isoprene in the rare adults is caused by the shared homozygous IDI2 variant (stop-gain mutation at c.431 position) driven functional aberrations of enzyme active site and metal–cofactor binding sites. Looking at the isoprene exhalation in our previous study 29, we assumed that the inheritance of the character (isoprene absence) has a recessive trait. Here, the heterozygous presence of the IDI2 c.431G > A variant in the isoprene deficient healthy parents and sibling sister of rare adult-1 and absence of this IDI2 mutation in unrelated healthy adults (isoprene normal) genetically confirmed our previous assumptions.
In this study, the prevalence (< 0.25%) of isoprene absent adults closely mirrored the actual homozygous prevalence (0.23%) of the IDI2 variant in the EU population whereas, the occurrence of isoprene deficient adults (64/1318 i.e. 4.8% among adults) did not closely mirror the actual heterozygous prevalence (6.95%) of the IDI2 variant in the EU population. This is mainly due to the fact that our clinical screening was not restricted to European subjects. The overall expression (homozygous and heterozygous) of the mutated IDI2 is different in other ethnic origins (Table S2) and therefore, the observed age distributions of isoprene, deficiency and/or absence may differ amongst another ethnicity/population. Besides, the cut-off limit of isoprene deficiency in adults was set by us to < 50 ppbV in this study. Exhaled isoprene concentrations can fluctuate by 5–25 ppbV in an individual simply due to his/her normal physiological variations in respiratory and hemodynamic parameters, natural menstrual rhythms and/or oral contraception and menopause 21, 33–35 etc.
While the expression of human IDI1 is conserved in various tissues and high within the mitochondria and proteasome of the hepatocytes, its divergent isoform IDI2 is highly expressed only within the peroxisome of the skeletal myocytes 36,37. Human IDI1 is poorly expressed in skeletal muscle. IDI1 and/or IDI2 catalyze the isomerization of isopentenyl diphosphate [(C14)IPP] to the highly nucleophilic dimethylallyl diphosphate [(C14)DMAPP]. In humans, the conversion of IPP to DMAPP takes place in two metabolic paths – during cholesterol biosynthesis in the endoplasmic reticulum of hepatocytes and during lipid catabolism (involving cholesterol metabolism) in the peroxisome of skeletal myocytes 38,39. Only DMAPP (not IPP) is converted to C5H8 via isoprene synthase (IspS) enzyme in plants 40–42. As humans do not have isoprene synthase and bioinformatic sequence alignment (whole exome/functional domain based) via BLAST search tool 43 did not locate human enzyme homologs of isoprene synthase, the wild type IDI1 and IDI2 genes (and related proteins) should serve as the determinant for human isoprene production from those two aforementioned metabolic paths.
Besides humans, while looking at other terrestrial and marine mammals, we observed interesting facts upon breath isoprene. In a recent pre-clinical study, mass-spectrometry based untargeted profiling of exhaled VOCs in spontaneously breathing awake healthy and/or influenza A virus infected pigs, we could not detect breath isoprene 44. On the other hand, in pre-clinical breathomic studies on goats and on cattle, we observed significant concentrations of breath isoprene from both ruminants 45,46. Via mass-spectrometry based comprehensive screening of exhaled metabolites from bottlenose dolphins, Aksenoy et al did not detect any trace of isoprene 47. Our present search in the Ensembl genome database 48 and EMBL-EBI resource 49 showed that IDI2 is not at all expressed in pigs and in bottlenose dolphins but is well expressed in goats and cattle, underlining functional IDI2 as discriminator between isoprene presence and absence. IDI1 is ubiquitously expressed in many tissues in all these animals.
Previously, via microextraction-coupled mass-spectrometric measurements of headspace of arterial and venous blood samples collected from mechanically ventilated humans and pigs, we observed extremely low (up to 10-fold lower than in human) isoprene concentrations within the portal and mixed venous blood of pigs 50. Such tiny fraction may be washed-out (i.e. stored previously) and/or produced via minimal IDI1 activity in the peripheral compartments. Nevertheless, as soon as the blood crossed the hepatic circulation, isoprene concentrations were diminished in hepatic venous samples – most likely due to a high rate of isoprene metabolism in pig liver. Similar to pigs and dolphins, only the IDI1 is expressed in rats and mainly within the liver. Most likely, due to a low (compared to higher mammals) isoprene oxidation rate in rat liver 51, Deneris et al had detected a certain fraction of isoprene in rat liver cytosol in vitro. They suggested that isoprene could be produced in rat liver via non-enzymatic degradation of IPP and/or DMAPP and postulated in general that breath isoprene is linked to hepatic cholesterogenesis 30. While the pre-clinical finding of Deneris et al was correct, the general inference drawn on the origin of human breath isoprene based on those outcomes from rats was wrong. In human hepatocellular microsomes, the complex cytochrome P450 enzyme system immediately oxidizes isoprene and isoprene monoepoxides to avoid hemiterpene toxicity 52. The oxidation rate in human liver microsomes is magnitudes higher compared to rats 51. Due to such high isoprene oxidation rate, hepatic cholesterogenesis is insufficient to contribute any considerable concentration of isoprene to human exhalation.
In the present study, despite normal plasma lipid profiles, bile substrates, sex-hormones and wild type IDI1 in rare adults and blood-related family members, significant aberrations in their isoprene exhalations confirmed our previously suggested 29 independence of human exhaled isoprene from hepatic cholesterogenesis and related principal pathways. Therefore, due to the absence of functional IDI2 expressions in these isoprene aberrated adults, the source of human breath isoprene should mainly be directly attributed to muscular metabolic activity and not to hepatic cholesterol biosynthesis.
Skeletal muscles represent around 40% of adult human body mass and predominantly utilize glucose and lipids to produce energy, regulate intramyocellular signaling and integrity 53,54. Insulin governs the balance between glucose and fatty acid metabolism in muscle 55 and peroxisomal beta-oxidation senses intracellular fatty acids and regulates lipolysis 56. Besides mitochondrial oxidation, peroxisomal beta-oxidation of very long-chain fatty acids, long-chain fatty acids and dicarboxylic acids produces acetyl-CoA. Acetyl-CoA is channeled towards farnesyl diphosphate (farnesyl-PP) production inside the peroxisomes 39. All enzymes (except 3-hydroxy-3-methylglutaryl-CoA reductase/HMGCR) step-wise converting acetyl-CoA to farnesyl-PP contain functional peroxisomal targeting signals and at the second last step of this pathway, IPP is converted to DMAPP via the IDI2 enzyme as only IDI2 is highly expressed here. Farnesyl-PP exits the peroxisomes to execute various metabolic processes in other cellular organelles.
Any kind of muscle movement/activity immediately gives rise to breath isoprene 11,57. Due to its low aqueous solubility and high volatility, isoprene is positively related to cardiac output and negatively related to minute ventilation 58,59. Both low-intensity and exhaustive exercise demonstrated an instant and profound increase in exhaled alveolar isoprene concentrations at the initial warm-up phase (that increases muscle perfusion) followed by gradual decrease with increasing work-load, which indicates its possible production and washout from the active muscle compartments 12,57,60. Exercise immediately increases skeletal muscular lipolysis, fatty acid transport from plasma to sarcoplasm and triglyceride hydrolysis to compensate energy demand. Thus, our present findings ascertain that isoprene is potentially originating from lipolysis in the skeletal muscle and wild type IDI2 denominates the presence of isoprene in exhaled human breath and also acts as the rate limiting factor for endogenous isoprene production.
Although we were able to detect the IDI2 mutation in PBMC, IDI2 gene and protein expression are skeletal muscle specific. Previously we observed IDI1 but not IDI2 gene expression in PBMC of healthy adults with absence, deficiency and normal breath isoprene 29. To assess the biological consequences of the IDI2 variant, gene and protein expression studies would require the collection of muscle biopsies of the affected adults. Those investigations are, however, behind the scope of the present study and limited by ethical considerations.
We discovered the genetic origin of human breath isoprene production and related biochemical routes. The rare character of isoprene absence/deficiency in healthy human adults is autosomal (10th chromosome, locus: 10p15.3) recessive. We translated isoprene as the first breath VOC biomarker with well-defined down-stream endogenous origin and metabolic pathways. This knowledge will redefine the clinical interpretations of this noninvasive biomarker for various physio-metabolic, pathophysiological and inherited conditions. We assume that the presence of DMAPP may not be essential for healthy human life as all principal pathways converting acetyl-CoA to farnesyl-PP are associated with lipid and cholesterol metabolism can utilize IPP to execute them normally. Already reported endocrine regulation 35 and age dependency 21,22,29 of isoprene exhalation indicates new research scopes of IDI2 activity in human aging, muscle mass development and related conditions. Similarly, further investigation of IDI2 gene and protein expressions in skeletal muscle tissue along with breath isoprene expressions under various exercise trainings and in individuals with muscle dystrophy and risk of rhabdomyolysis e.g. under statin interventions or injury may reveal unexplored frontiers in sports/fitness and musculoskeletal medicine and inter-organ metabolic cross-talk.
In order to understand the evolutionary significance of human IDI2 gene and the presence/rationales of isoprene production pathway in human, we need further multi-omic based system-wide evaluation from the genome to up-stream cascades. Tissue specific gene and protein expression followed by transcriptomics and proteomics may lead us to the actual enzymatic and metabolic significances (DMAPP to isoprene) that are linked to the last IDI2 exaptation taken place ~ 70 million years ago for an unknown function. Inducible IDI2 deficiency in animal models could also shed light on the kinetics and physiological background of isoprene metabolism. Further investigations should screen a large number of isoprene deficient subjects for IDI2 allele frequencies to realize its exact genetic correlation(/predisposition) factor with breath isoprene expressions. In order to apply isoprene or any other endogenous biomarker to routine clinical practice, well-defined down-stream origins and pathways should be addressed.