According to the International Osteoporosis Foundation, globally, one-third of women aged >50 years experiences osteoporotic fractures. Presently, there is a significant diagnostic gap that hinders timely therapeutic intervention to prevent fractures. Here, for the first time, we have investigated associations between exhaled volatile metabolites, serum bone markers and the risk of osteoporosis assessed by quantitative bone density index.

Before the beginning: At that time, Margaret and I were living in an apartment in Rostock. One evening during the late summer 2016, I was reanalysing research data from five subsequent clinical studies. These were conducted on healthy humans to understand a series of physiological effects onto exhaled volatile metabolites. For obvious reason, Margaret was amongst a few common participants in all those studies. While looking at her breath data from the steady states of spontaneous breathing, I observed pronouncedly varying concentration ranges of endogenous metabolites. Nevertheless, her two data points measured in two different months from 2015 were almost reproducible. Surprisingly, this was not the case for my breath patterns. I looked further at the breath data from our research group members from those studies and similar fluctuations were observed only in female colleagues. At dinner, I told Margaret about her strange breath patterns and that instantly poked her incorrigible curiosity. It is noteworthy that she is not a scientist by profession. However, she started to suggest various possibilities starting from her mood, meals, work stress etc. on different sampling days and all of a sudden, she placed her period calendar on the table and said, “Aha! – I think that my monthly cycles have something to do with this – what do you think?”. I decided to investigate this in detail after seeing an interesting fact that those two reproducible datapoints did actually coincide with her two independent menstrual bleeding phases. Within next three months, it turned out that her wild intuition was worth pursuing. Perhaps, that’s why science should be communicated to the society via non-expert summaries and/or stories such as, Behind the Paper.    

At the beginning: After having the ethical approval, young and healthy females (aged between 18 – 45 years) were invited to participate in an observational clinical study. Besides seeing the endocrine effects of natural menstrual rhythms on exhaled metabolites, it was surprising to observe an overall suppression of potentially endogenous volatiles exhaled by women who had undertaken daily oral contraceptive pills. In 2018, we published these findings in Scientific Reports¹.

We have realised that the constant supply of synthetic sex hormone(s) via oral pills disrupts the natural endocrine homeostasis and thereby, diversely affects breath profiles. Thus, I was interested to see what happens in women after menopause. Given the fact that the oestrogen hormone starts to decline naturally after menopause and that predominantly affects the bone mineral homeostasis, I was explicitly interested in examining postmenopausal bone metabolism via breathomics.  

Consequently, women (interested colleagues, friends and family members) aged over 50 years were invited to volunteer our initial pilot measurements. During participation, they were asked for signed consents and to disclose details of any existing clinical condition (especially on bone health) that they might have. One of those mornings, Prof. Fischer from the Dept. of Pediatrics walked into our mass-spectrometry lab in response to my invitation to participate. After participation she said that my idea is unique and extremely interesting. She has an existing cooperation with Prof. Mittlmeier from the Dept. of Traumatology, Hand and Reconstructive Surgery, who has patients of my present interest. If I want, then she can arrange a meeting to discuss further research scopes.

Hereon, things started to take a bigger dimension. 

After the beginning: During the next week, my doctor-father (Prof. Schubert), our analytical science lead (Dr. Meikisch) and myself met with Prof. Fischer and Prof. Mittlmeier. Soon enough, a research proposal was planned for the German Research Foundation (DFG) grant application with the aim to investigate postmenopausal bone metabolism and osteoporosis via breathomics. In 2018, we submitted the proposal and after the second round of peer-review, it was rejected by the DFG. One of the reviewers was not convinced that effects of bone metabolism can reflect in exhaled breath. At that time, there was no supporting literature available on breath-bone cross-talk.

While I was very upset, Prof. Schubert, Prof. Fischer and Prof. Mittlmeier assured me that the study can still initiate and some remaining funds from other concluded projects could be used to execute this important and promising campaign in a reasonable and clinically relevant scale. Although the consumables and other experimental costs were covered, my post-doctoral position as the principal investigator was at risk. At the 11^th hour, our clinic director (Prof. Reuter) came to the final rescue and extended my employment for 24 months (which was temporary back then). We were all set to explore!

Two highly motivated medical students (Mr. Grzegorzewski and Miss. Broderius) were assigned to include postmenopausal women into two consecutive but independent clinical screenings for the biomarker discovery and validation, respectively. We were fortunate to have a dedicated lab-technician (Ms. Rahn) at the Dept. of Pediatrics, who took care of the detailed and timely analysis of the serum bone markers. 

Until late 2018, a discovery cohort of 127 postmenopausal women were examined to find breath volatile markers and quantitative cutoff levels for osteoporosis. In 2022, a part of this discovery data was published in iScience, where we described physio-metabolic effects of healthy female aging and menopause on breath biomarkers². Meanwhile, with an unexpected delay of seven months due to the COVID-19 pandemic, until early 2020, an independent validation cohort of 52 women were recruited and repeatedly followed up for reproducibility and seasonal effects. Our interdisciplinary expertise enhanced further as Prof.  Kreikemeyer (Institute of Medical Microbiology, Virology and Hygiene) joined the consortium.

End of the beginning: In 2024, we discovered and published in Communications Medicine that predominately gut-originated metabolites are strongly associated with postmenopausal bone health³. These results passed the independent validation along with repeated follow-ups during seasonal changes. Our non-invasive breath test offered a more rapid and reliable risk classification tool for postmenopausal osteoporosis than the conventional serum markers.

These findings elucidated breath markers of bone metabolism via gut-bone axis and enabled personalised and point-of-care assessment of osteoporosis risk and paths towards novel and yet unexplored therapeutic targets.

Now as then, we are all set to explore!

As Winston Churchill once said, “Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning.”

References:

1. Sukul, P., Schubert, J. K., Trefz, P. & Miekisch, W. Natural menstrual rhythm and oral contraception diversely affect exhaled breath compositions. Scientific Reports 8, 10838 (2018).
2. Sukul, P. et al. Physiological and metabolic effects of healthy female aging on exhaled breath biomarkers. iScience 25, 103739 (2022).
3. Sukul, P. et al. Exhaled breath metabolites reveal postmenopausal gut-bone cross-talk and non-invasive markers for osteoporosis. Commun Med 4, 1–14 (2024).