The featured fundamental discovery of the April 2021 Special Issue of THE INDIAN ROVER:
Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin (P. Sukul et
al. 2021, Cell-Press: https://www.cell.com/heliyon/fulltext/S2405-8440(21)00027-X)
Scientists/Authors & Affiliation:
– Dr. Pritam Sukul, Senior Medical Scientist, Dpt. of Anesthesiology and Intensive Care, University
Medicine Rostock, Germany.
– Dr. Anna Richter, Medical Biotechnologist, Dept. of Hematology, Oncology and Palliative
Medicine, University Medicine Rostock, Germany.
– Prof. Dr. Jochen K Schubert, Deputy Director, Dpt. of Anesthesiology and Intensive Care,
University Medicine Rostock, Germany.
– Dr. Wolfram Miekisch, Head Analytical Chemist of ROMBAT group, Dpt. of Anesthesiology and
Intensive Care, University Medicine Rostock, German
Dr. Pritam Sukul (DSc. Medical Sciences) In Conversation With Tirtha, Editor-In-Chief, The Indian Rover
Tirtha: Jay Gour, Pritam da! Greetings from The Indian Rover! Although we share an invisible bond from spiritual fraternity, motherland and mother-tongue, today I am sent by the editorial board to conduct the interview, representing our magazine. So, I must admit it’s a double joy from my side as I congratulate you & your team on the excellent discovery in the field of medical science.
Pritam: Jay Gour, Tirtha! Good evening from Germany! Well, there’s certainly no doubt that The Indian Rover has summoned the correct person to greet and interview me. First of all, thanks to my motherland’s biggest literary magazine and to you for your kind interest in our recent research findings. We are truly honored to be featured in this special edition.
Tirtha: So, before embarking to the research segment, I am curious to know whether the life of a foodie Indian scientist in Germany is jovial or banal.
Pritam: Ha ha! Good one to start the conversation! In my humble opinion, your inquiry sounds rather Freudian. To be precise, I have been to many places on earth for study or research, and due to my incorrigible Indian food habit, it’s hard to assume a place abroad where I haven’t faced challenges. Missing Mom’s kitchen is not funny! Nonetheless, I haven’t witnessed a better place for independent and co-operative research than Germany. Notably, the ROMBAT research group (especially, my mentors, Jochen and Wolfram, and my colleague, Patricia) always stood by me as a family since my arrival. Despite many ups and downs in my personal and professional life, they never left me alone.
Now, it’s my second home and I love this country and its people. So, for me it’s undoubtedly jovial.
Tirtha: Well, talking of research: you have been awarded with the prestigious Marie Curie Fellowship, attained a hat-trick publication in the Nature Research, and you have just published the seven years long clinical study conducted upon more than 1000 humans in the esteemed Cell-Press & ELSEVIER-ScienceDirect. As you often say that science is a joint effort, I am willing to know what role have your colleagues, especially Dr. Schubert and Dr. Miekisch, played in establishing you as who you are today?
Pritam: Yes absolutely! As we are doing interdisciplinary research, involving physiology and medicine, with analytical chemistry and bio-technology, it must be a joint effort. Therefore, our ROMBAT group is composed of clinical and analytical scientists, physicians, chemists, biotechnologist, engineers etc. and Prof. Schubert and Dr. Miekisch are among the pioneers in the field with over 20 years of experience in basic, pre-clinical, and clinical investigations with regard to applications of Breathomics and volatile Metabolomics. As I have mentioned earlier, no matter what I am today or may become in the future, these two persons have always been my best supportive shields. They have trained me to conduct complex and cross-disciplinary research as well as supported me to balance a personal life in parallel. They took me from a ‘small town’ of India, when I had no experience in Breathomics except some basic knowledge, ideas and mainly motivation. This is not only in my case! Many a times, I have seen them selecting young individuals, depending upon their ‘research motivation and interest’ and not explicitly based on the academic CV or prior experiences or number of publications etc.
Tirtha: This paper, titled as ‘Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin’, which you ascribe as a “fundamental discovery”, has caught our attention. I will be pleased to know the background and what it actually states for non-experts. It will be greatly appreciated if you can answer in a lucid way, avoiding scientific jargons as much as possible.
Pritam: As you have rightly mentioned, this is a fundamental discovery in medical Breathomics. At first, let me tell you a bit about Breath Analysis. A very tiny fraction, less than one percent, of the human breath is composed of thousands of volatile organic compounds called VOCs. VOCs are produced within our body due to biochemical pathways, metabolic processes (at cellular or organ level) as well as physiological and patho-physiological conditions or under certain administered therapy. Afterwards, these VOCs are transported via blood to the lung and exhaled through our breath. We are identifying and quantifying these organic substances in order to achieve non-invasive and rapid detection of diseases and monitoring of physiology and metabolism or therapy.
Among those VOCs, isoprene (C5H8) is the second most abundant, endogenous (i.e. produced within our body) and clinically the most important biomarker (concentration range in adults: around 100 – 300 ppbV). Until now we believed it to be the prominent by-product of the Mevalonate pathway (i.e. Cholesterol Biosynthesis), where mevalonic acid is deduced to non-sterol isoprenoid molecules. This endogenous metabolic origin was hypothesized by Deneris and colleagues in 1984 via pilot in vitro observation on rat liver cell cultures and since then it is theorized as the rule of thumb and is widely believed in clinical science, medical metabolomics and breathomics in order to interpret experimental data. Hundreds of clinical studies have repeatedly reported and published the differential expressions (i.e. observed between healthy and sick groups) of exhaled breath isoprene concentrations as promising non-invasive breath biomarker to detect/monitor diseases such as cancers, lung diseases (COPD, asthma, fibrosis etc.), myocardial infection, hypercholesterolemia, oxidative stress, chronic liver disease and many other health conditions. Moreover, relative changes in exhaled isoprene concentrations mirror various physiological and metabolic effects (P. Sukul et. al. 2016, 2018 & 2020. Nature Research Portfolio). According to published research in The New England Journal of Medicine, absence of non-sterol isoprene is attributed to inherited errors (e.g. hyper immunoglobulinemia D syndrome, auto-inflammatory periodic fever syndrome and mevalonic aciduria) in mevalonate pathway. Breath isoprene deficiency in Duchenne muscle dystrophy patients supports its storage and washout from the muscle compartments. Despite all those mind bobbling findings, isoprene couldn’t enter to routine clinical practice as a definitive diagnostic marker because many other experimental observations upon breath isoprene expression remained unrelated to the corresponding patho-physiological effects (i.e. well known) on its putative metabolic origin (i.e. Cholesterol biosynthesis). In spite of gradually growing doubts upon the conventional origin of isoprene, none could prove it wrong or find the truth. Therefore, such an excellent biomarker remained shrouded in mysteries until our present discovery.
Tirtha: So, what does it disqualify?
Pritam: We have disqualified an important theory in clinical metabolomics and breathomics, which was believed since last 36 years; I mean, the putative origin of isoprene from Cholesterol Biosynthesis as was hypothesized by Deneris et al; around a year before my birth.
Tirtha: So, what are your assumptions regarding how beneficial it can prove to medical science, and above all, human health?
Pritam: These findings will re-address our state-of-the-art knowledge and understanding upon the
effects of different types of cancers, cardio-pulmonary diseases, hyper-cholesteremia,
oxidative stress, neurodegenerative disorders as well as certain rare inborn genetic errors
onto this most important and omnipresent exhaled breath volatile- isoprene. This will redefine and rationalize the clinical interpretations of isoprene as biomarker towards the rapid, non-invasive assessment of various physiological, metabolic and pathological conditions in
real-time via breath analysis.
Tirtha: As medical science generally frames a hypothesis first and then examines it, when did you first conjectured the hypothesis of the paper?
Pritam: In May 2013, I was awarded with the esteemed Marie-Curie Fellowship on my ideas to conduct intensive research in Experimental Anesthesiology and Clinical Breathomics at the University of Rostock (Germany), under the eminent supervision of Prof. Dr. Schubert and Dr. Miekisch. In few months, I had recognized the fundamental problem with physiological and metabolic confounders, which often override the actual pathological information in breath biomarkers. Consequently, I stumbled upon the believed origin of isoprene, while explaining those effects. I came up with the initial idea that the key to this mystery is hidden in the healthy adult humans, who are at the lower extremities (i.e. significant deficiency) of exhaled isoprene concentrations.
Tirtha: What were the odds that were there in the path & how did you overcome it?
Pritam: Indeed, there were obvious challenges or odds. It is very unlikely to find healthy adults with isoprene deficiency (because it is already a characteristic of inborn metabolic disorders and diseases as mentioned above). According to statistical power test we need to investigate a sample size of at least 1000 humans which is almost a day-dream in academic research, especially the ones that are conducted in universities. To conduct such a clinical study, we need to invest at least 5 to 7 years of unprecedented efforts and huge expanses. The chance of failure is still almost 99% as we may not find any healthy adults with isoprene deficiency. Finally, I have to convince my mentors that I am crazy enough to do it.
To be honest, I did not have any good idea or plan to overcome those at the first place, but I was ready to confront. My mind simply relied upon the early teachings of my late grandfather from the Śrīmad Bhāgavad Gītā: “karmaṇye vādhikāraste mā faleṣu kadācana” – your rights are only on the deed, not on its rewards – Lord Kṛṣṇa’s advice to Arjuna, before the Kurukṣetra War. I know that may sound philosophical, but I do have no better response.
Tirtha: Well, Bhāgavad Gītā, is undeniably the biggest source of motivation any human being can find! So, what happened then when you proposed this idea?
Pritam: On a fine Friday morning lab-meeting, I presented the cumulative ideas to Prof. Schubert and Dr. Meikisch. After a span of absolute silence in the room, I heard the most awaited response from Prof. Schubert: “Go ahead! We are with you. No risk, no science”. Dr. Miekisch with a similar response added, “Let’s do this. Even if we fail, that would still be a substantial contribution to science”. On the same day, three of us conceptualized and designed the large study(s) to investigate a series of physio-metabolic effects and with the hope to address that fundamental problem of isoprene origin. We started the experiments soon after having the ethical approval from the Institutional Ethics Committee (IEC) of University Medicine Rostock.
Since 2013, I have identified and quantified hundreds of VOCs via high-resolution mass-spectrometry based clinical breathomics within the exhaled breaths of human subjects, who belong to different age, gender, health conditions and ethnicity. We applied various physiological manoeuvres — respiratory and hemodynamic – to observe immediate effects on isoprene and other VOCs. We used the advanced Proton-Transfer-Reaction-Time-of-Flight-Mass-Spectrometry (PTR-ToF-MS) to monitor the instant behaviour of isoprene, breath-by-breath in real-time. Nevertheless, until 2015, we had no luck upon the occurrence of any isoprene deficient adult.
But in October 2015, I have received “The Gift of Mother Nature”.
Tirtha: “No risk, no science” — what a boisterous prophecy in what brief words! I’m compelled to admire Dr. Schubert’s genius!
Pritam: I actually regard him as my ‘Guru’ in science. He is the Deputy Director of the Dept. of Anesthesiology and Intensive Care of our University Medicine Rostock (UMR). Prof. Schubert is a senior anesthesiologist and chemist with unparalleled knowledge and understanding of Breathomics. He is our group leader and I have rarely seen a wise person as he is. For me, he is a father figure. During this afternoon, I had informed him regarding this tentative interview and asked if he wants to be present. His immediate reply was, “you are the first author and it’s your work so go ahead”.
Tirtha: That’s so generous of him! Please do also enlighten us about Dr. Miekisch’s visions as well about the contribution of Dr. Richter.
Pritam: Dr. Miekisch is the Head of the Mass-Spectrometry Laboratories at the UMR. He is an analytical chemist with over 20 years of experience in applied bio-analytical research he is the principal analytical chemist of the ROMBAT group and is leading all bio-analytical research activities involving pre-clinical and clinical breathomics. He is responsible for analytical setups in our clinical studies such as studies on severe lung diseases, cancer, metabolic changes or drug monitoring. He has contributed to the present state-of-the-art in breath sampling and analysis with around 100 publications. Currently he is the chairperson of the International Association of Breath Research (IABR) and leader of the IABR task force for sampling and standardization.
We are also utterly grateful to Dr. Richter as she had performed the tedious genetic analysis for this study. She is an excellent and enthusiastic young woman in medical science, who collaborated with us in this study and helped us to obtain all the genetic data and corresponding interpretations on time.
Tirtha: As you told me that you have received “The Gift of Mother Nature”, what do you imply?
Pritam: I call it, an important life event! On a Wednesday morning at around 10:30 am, a young and healthy lady walked into our lab to participate in the ongoing study. As soon as she started breathing into our instrument, the exhaled isoprene signal completely vanished from the monitor and the isoprene curve at the mass scale was flattened to zero. I was surprised and worried as I thought that I have somehow crashed the expensive mass-spectrometer. I must rapier that as soon as possible. I rebooted the instrument, recalibrated the entire mass scale and I immediately breathed into the sampler, myself. Thank god! The isoprene signal and its mass-scale curve were resumed. But, as soon as she re-breathes into the instrument, isoprene signal and mass completely disappeared once again.
Eventually, I realized that Mother Nature has just gifted me with the key to a fundamental problem. We have found a healthy adult not only with long-anticipated deficiency but complete absence of breath isoprene. She is living a normal life without any acute or chronic diseases. That’s utterly astonishing!
Tirtha: Very interesting and intriguing! What happened next? What have you done with this wonder?
Pritam: I had an exciting meeting on the same afternoon with my mentors. They told me that colleagues in the UK and Innsbruck had encountered 2 to 3 such cases (with very low isoprene) during the last decade but no down-stream investigation was performed to date. Thus, we decided to call upon the immediate blood-related family members of our ‘subject without isoprene’. Both of her parents and sibling sister turned out to have significant isoprene deficiency. Nevertheless, all of them are also healthy and do not suffer from any known consequence of this pathological phenotype.
Tirtha: What are the eventual findings, the thing that science is always hungry for? Which actions did you take?
Pritam: In my humble opinion, anything that is fundamental, philanthropic and substantial do strive science. In this case, we had few key questions. Do they have a different/rare kind blood cholesterol and/or lipid-profile? Is any of the regulating genes of the Mevalonate and Cholesterol pathway enzymes not expressed in this family? Is there any over or under expression of those regulating genes? If nothing is abnormal, – is the putative origin of isoprene in human is misunderstood? Therefore, we performed blood analysis for cholesterol and lipid profiling, hemodynamic monitoring and genetic analysis of the subject without isoprene and her isoprene deficient family members. All genes of the mevalonate pathway enzymes were analyzed. In parallel, we included other healthy unrelated adults with normal breath isoprene levels to our experiment. As menstrual cycle in women is known to affect exhaled isoprene concentration (P. Sukul et al. 2018. Nature Research Portfolio), we had measured the ‘lady without isoprene’ throughout her monthly cycle. In 2018, while she was pregnant and we measured her breath throughout her pregnancy and after child birth in search any accidental or incidental appearance of isoprene in her breath. We measured her young and healthy husband (not-blood related individual and with normal isoprene levels (i.e. >100 ppbV) and her new-born daughter (also without traceable isoprene).
Tirtha: I’ll be glad if you can briefly narrate the outcomes of the research for our readers.
Pritam: The outcome was astonishing. In contrast to conventional concept, we witnessed that adults can live healthy without exhaling isoprene or with significant deficiency. This rare phenotype represents a recessive genetic inheritance. Despite physio-metabolic changes during menstrual cycle are well-known to profoundly affect isoprene exhalation, profoundly increased plasma cholesterol during pregnancy and after childbirth, isoprene remained absent. All genes of mevalonate pathway enzymes were normally expressed in all participants, without any down-regulation or compensatory up-regulation. The putative metabolic origin of isoprene turned out to be misleading. Till date all published studies (including our own) in clinical breathomics have misinterpreted this important biomarker due to a wrong fundamental concept. Absence/suppression of true isoprene production process in our healthy subject and family have summoned further studies to track the possible presence of any compensatory mechanism in such individuals. Clinical translation of our basic findings will offer an unexplored frontier towards the limited clinical understanding of certain ultra-rare and inborn genetic diseases e.g. Mevalonic Aciduria, Muscle Dystrophy etc. Our upcoming study will address the correct metabolic origin of isoprene and will trail-blaze the rational clinical interpretations of isoprene exhalation under various health conditions such as cancer, cardio-pulmonary and liver diseases, neuro-degenerative disorders, as well as, in viral infections and viro-bacterial co-infections. Our present findings have refined and optimized the clinical interpretation of isoprene as biomarker in medical Breathomics.
Tirtha: Now, as science is rooted in its fight against stagnancy, what have you planned about your future endeavours?
Pritam: Well, science is not a one-day cricket or a football match. Science runs slow but steady. I have eventually learnt to live and grow with it. I have dedicated myself to science and will do so until I breathe my last. My future goal is to build trans-national networks of research between India and Europe and bring young minds and talents into action.
Tirtha: You have been a seminal figure in the present Indo-German ties on medical research. What future so you envisage for these two countries that are built by the āryas?
Pritam: I don’t know, if I am so seminal but I do enjoy working in trans-national research and innovation frameworks. I have gained our two precedent. It is not hard to imagine that the future of science demands the bilateral cooperation between the land of ideas, science and innovation (Germany) with the land of philosophy and spirituality (India). Personally, I regard philosophy as the soul of science and thus, a cordial culmination of both will unequivocally foster the advancement in search of truth towards the betterment of mankind.
Tirtha: Whom do you wish to thank for all these trail-blazing discoveries? Whose whispers of love solace the dry heart of a scientist?
Pritam: I must thank Jochen and Wolfram along with my parents (Maa & Baba) and immediate family members (Babi, Jethu & Kaka), teachers and friends for their trust, teaching, love and support. I do dedicate this to my Maa, Motherland, Mentors, Margaret, and Mahāprabhu.
Now, I must be very careful to answer your second question, in order to avoid any minimal chance of domestic violence.. Ha ha! In Sanātana dharma, the equal importance is given to women (or wife) in relation to the success and even spiritual enlightenment– the Śiva-Śakti embodiment. I am blessed to have Margaret who has been the quantum of energy and the oasis of love on the sands of my time.
Tirtha: What role does the Indian culture, especially the tenets of Sanātana dharma, play in your research & life?
Pritam: Given the fact that I took birth in a Brāhmaṇa family at the very birthplace of Śrī Caitanya Mahāprabhu, dhārmik acts and spiritual practices have been obligatory attributes of my life. As I had mentioned earlier, Bhāgavad Gītā empowers and leads my mind during chaotic situations. Similarly, daily practice of meditation and ritual gestures help me to control my thought process and enrich concentration, which are indispensable pre-requisites for science and research.
Tirtha: Thank you, Pritam da, for such a candid & enlightening talk. I have always opined, just like the sages and ṛṣis of the past, that Sanātana is Science, and Science is Sanātana. It is indeed bewildering to observe that you are carrying forward the legacy of the great sons Bhārata Mātā has produced. May thy name be immortalized like Caraka and Suśruta and may Lord Dhanvantari lead you to more astonishing discoveries in the field of medical science. To imitate you with a little inclusion of mine: ‘Cheers to the Sanātana Science for Life!’
Words of the editorial board on Dr. Pritam Sukul et al.:
Based on independent nominations, recommendations and community comments from the national and international scientific experts, journal editors and forums (Government and Public Bodies), we have decided to feature a latest fundamental finding in clinical science, entitled as ‘Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin’, contributed by a young NRI scientist named Dr. Pritam Sukul and his German workmates (viz. Dr. Anna Richter, Prof. Dr. Jochen K. Schubert, and Dr. Wolfram Miekisch of the Rostock University Medical Centre, Germany).
Our editorial team has studied the aforementioned article, which is published in the prestigious Cell Press, Science Direct, Elsevier and Research Square (pre-print server) along with Dr. Sukul’s other original works in the field of medical Breathomics & volatile Metabolomics (available at the Nature Research, IOP-Science, MDPI etc.). We have found those as a remarkable stack of contributions, depicting continuous advances.
The featured research article links:
ELSEVIER ScienceDirect: https://www.sciencedirect.com/science/article/pii/S240584402100027X
Research Square: https://www.researchsquare.com/article/rs-140360/v2