What if scientists made a further substantial contribution to the fountain of youth? How isocitrate extents the life-span of cells and how new synthesis approaches make this compound available on large scale

TABLE OF CON TEN TS 1. 2. 3. 4. 5. Executive Summary Intro: Science discovers the fountain of youth Compounds and their impact on the youth of cells Isocitric Acid (ICA) – the “hidden gem” Problems with bulk sourcing of isocitrate - why is isocitrate not as prominent as the other compounds? 6. Innovative/ new approaches to Isocitrate – Making the compound available on larger scale 7. The great future of isocitrate Navigatethroughthiswhitepaperbyclickingontheheadlines/chapters!

EXECUTIVE SUMMARY For human beings there is a “natural, intrinsic” age of about 120 years that could be reached without the new and still premature approaches of gene editing and stem cell technology. Key to a long life is the “young, healthy cell” rather than curing single diseases. compounds, some natural from a healthy life style, also the supplement/ Apart administration of especially members of the so called alpha hydroxyl carboxylic acids, were proven to support cells youth. One prominent member of this compound class, isocitric acid, could not yet unfold its respective potential because of lacking economically feasible production processes. This bottleneck was removed by several research institutions and companies only recently. Now, one can expect many new vitalizing / juvenescent products potentially increasing our life-span that are based on isocitric acid with several application fields in pharma, cosmetics and food industry.

I n tro: Sci en ce di scovers th e fou n tai n of you th As everything else in mythology, the “fountain of youth” is just a vague, allegoric object that no living being has ever seen in reality. Nevertheless, as with most such myths, the search for - and the believe in - this very nebulous spring has never come to an end. Quite the opposite is true: The desire to become young again – or, even better – not to age at all, has been fuelling thoughts, art, research and activities for centuries. Meanwhile, many aspects of science on the one hand and of dreams and mythology on the other hand have found to share common features and to form intersections. So, especially the discovery of the very nature of ageing opened a deeper understanding of what (theoretically) needs to be done to prevent early dying. The individual age, hence the state of being “young” is not invariably marked with a burning candle somewhere in the afterworld. The status of maturity is inscribed in every cell of a living being. The game changing point is that this status can not just been read but it can be influenced and changed! Sounds great – but how to “keep cells young”? If you look around in the faces of people, you could get the feeling that – so far - not much has been accomplished in this field. This impression is not really true. Just compare today’s faces with those of our grandparents of same (numerical) age. Such comparison of appearance, of course, is no scientific approach. However, it already gives you an unambiguous impression. the So, what (if any) has already been changed in ageing? What still could be done? Basically, there are three main strategies to prevent ageing in its biological sense: a) b) c) Delay ageing of original, existing (healthy) cells Replace old / dying cells by new ones Change the DNA code of cells Naturally, this is not THAT easy, but many doors have already been opened, some approaches did already prove to work and a few of them have already procedures, viable treatments and business models. turned into Perhaps the most important discovery was, that - in fact – trying to cure the many individual (chronic) diseases by numerous artificial drugs with all their unwanted side-effects is a dead end strategy for the goal to keep people healthy, fit and long-lived. As most diseases are just the consequence of getting older, of developing a weaker immune system, less powerful organs and worn out tissues, YOUTH is key to address this matter! So, if one managed to keep cells as young as possible, most diseases never would occur and, consequently, no treatment would be needed [1]. As we know, points b) and c) are still far from being tools of everybody’s everyday’s life. Regardless of the breathtaking progress in these fields, stem cell technology and gene editing still need to be further developed as to become viable means of rejuvenating human beings. So, every single step that has already been taken towards longer youth in the past belongs to category a). Considering this, the results are encouraging and surprising. The average life span in the U.S. has increased by about 16% during the last 50 years:

Life Expectancy 1950-2021 in the U.S. (<ahref='https://www.macrotrends.net/countries/USA/united-states/life-expectancy'>U.S. PIC.: Expectancy 1950-2021</a>) Life figure: reflected by another As we know, part of this result is based on lowering the infant mortality, on developing vaccines against prominent infectious diseases. The main contribution, however, originates from “younger, fitter cells” of adult / older people. This is better the percentage of the US population being 64+ years old, the so called “elderly dependency ratio”. It grew from 15.1% in 1960 to 24.1% in 2018 – a rise by about 60%! [2] Even if one considers today’s lower birth rate (it dropped from 24,200 to 12,000 per 10,000 people in the same time span), that also contributes to an increase in the elderly dependency ratio, this number is really impressive. The people do not just live longer, they also are healthier. What exactly made the cells “younger” during the last decades? Again, there are two sides to be considered: reducing (negative) “stress” on the one hand and actively supporting cells on the other hand. At the beginning – back at the early 20th century, when nobody knew much on the ageing processes, only the first aspect – more or less unintentionally - unfolded its power: The cell stress declined by the general increase of the living standard: Less hard work, less dangerous working conditions, much lower toxin levels in food, houses and drinking water, lower tobacco consumption, less alcohol, less polluted air, water and ground, sufficient food, more holidays, relaxation opportunities and many more other things … All these aspects contributed not only to the individual feeling of “well-being” but they also prevented our smallest living building block, the cell, from ageing too fast. The second point, actively catering the cells’ needs, only came into our focus when we had discovered the main mechanisms of these “elements of life” – at some time in the 1970ies and 1980ies. We did not only learn to understand the complex processes of cell division, of DNA- and RNA replication, of cell death (apoptosis) and of factors that transform a normal cell into a cancer cell – but we also got insight into the matter and energy transforming processes of these astonishing masterpieces of live.

that And exactly here, some measures to support these processes have already been found - but, on the other hand, also much potential still is waiting for exploitation. Even someone not explicitly educated in biochemistry, biology or microbiology knows such biochemical intracellular processes definitely depend on factors such as the availability of starting materials, (enzymes) and energy. Additionally, transport systems and information cascades are expected to be of importance. So, beyond the hot topics “gene editing” and “stem cell technology” there are thousands of factors that could be used to influence cell metabolism and, consequently, the process of cell ageing! What exactly can be done? catalysts One could classify the approaches into two main groups: • • Applying / furnishing compounds (and energy plus stimulation signals) that are rare in cells but heavily needed that are toxic, Diverting compounds harmful, present in levels above the tolerable level; avoiding too much stimuli, too much energy in some aspects really hostile: Sounds logical, but is not that easy to accomplish in reality. We have learnt that many (negative) stress factor levels could be lowered in the course of the last decades. That results in a better availability of cell nutrients and – to some degree – also in a better removal of harmful by-products. On the other hand, the natural environment of the human being became more and more artificial, our organism, our cells do not really live under the conditions they had been designed for any more. So today’s picture resembles a farmer who was fed up with all the many bad harvests in the open field and flatfooted decided to grow all his plants in an over fertilized greenhouse with carbon dioxide ambient temperature, plenty of water, pesticides and 24 hours artificial illumination. He eliminated all the formerly limiting factors in good faith but ended up with new problems and hardly more edible, tasty fruits. This exactly is the paradox situation: We could eat all the necessary food – but we consume too much of the wrong things; we are able to address atmosphere, always

all the normal stress factors – but we did not just eliminate them but replaced them by new ones. And this, in turn, has a distinct impact on our health and life-span as well. Since a couple of years, the average youth’s life expectancy in the U.S. stagnates (refer to the graph above) – a first time situation since the 18th century! It is now well recognized that human beings could reach an average age of 120 years at a relatively healthy condition WITHOUT gene editing and stem cell technology. This is not just a guess but a figure derived from studying the cell processes and cells’ inherent capacity of renewal and repair [3]. So, why does the average individual hardly reach their 100th day of birth? It is all about discrepancies between real living conditions and ideal ones. If we consider the two main factors again – sufficient and information on the one hand and low levels of toxins, surplus compounds and negative stimuli on the other hand – one becomes aware of problems that prevent us from growing that old: (Ref. to PIC.: Prolems that prevent us from getting 100+ years). nutrients, energy, stimuli All these facts are not really new. They have been paving ground for thousands of publications, research programs and strategies on extending life-expectancy (without addressing genes or stem cells directly) during the last 30 to 40 years. They also form the basis of a multi billion $ industry providing either drugs, nutrients, supplements or services in the field of staying young. The most basic – but nonetheless very effective – approach is that of sports and physical activity. It not just compensates parts of our “artificial inactivity” tissue functional but it also balances the levels of nutrients, stimuli and toxins on the cellular level. underused keeping by Again, people tend to exaggerate here as well. The beneficial effects can not be increased by just adding some extra miles to one’s weekly jogging plan. About 2-3 hours of intense activity a week are the optimum and beyond that – there is more harm than good…[4]. Sufficientnutrients,energy,stimuliNotjust“enough”butmuchmoreenergythanrequiredAwrongcompositionoffoodlackingmanymicronutrientsLackingstimuliresultingfromsunlight(vitaminD),weather(temperaturevariations)andphysicaleffortsAdditionalcellstressinducedbyconstantlyhighlevelsofsugarandfatcomponentsLowlevelsofundesired,toxiccompoundsandenvironmentalfactorsHighconcentrationsof(toxic)metabolicproductsasresultofthe“overfeeding”Additionalartificialtoxinsresultingfromchemicals,artificialmaterials,non-naturalelectromagneticfields(X-ray,microwaves,magneticfields)lackoffreshair,(water),silenceanddarkness(duringnights)toomanypsychicalstressfactorsincludingirregularrhythmsofactivityversusin-activityresultinginnegativestressPIC.:PROBLEMSTHATPREVENTUSFROMGETTING100+YEARSBYCHRIOBLOCK

Compou n ds an d th ei r i mpact on th e you th of cells What else? Everybody knows practical forms of such “cell supporting” activities besides sports: there is the food supplement industry with vitamins and other beneficial agents, there is cosmetics with “cell-enhancing” ingredients from hyalurone to aloe vera, there are “detox” strategies and products. the all Not all of these approaches are scientifically based and really effective. Some of them, however, are. The most visible effects can be reached with our skin, hence with “functional cosmetics”. There is no doubt that special agents / ingredients of cosmetics benefit the skin cells – both in protecting (UV-blockers) as well as in a regenerating (vitamins, nutritions) manner. Less visible, but nevertheless existent, there are also proven strategies to cater for the cells of other tissues by providing micro-nutritients. Two prominent examples are vitamin D (stabilizing the entire immune system) and omega-3 fatty CITRIC ACID CYCLE acids. Numerous studies have been published giving evidence that a regular up-take avoids diseases and, hence, prolongs life [5,6]. Other promising compounds are the so called “alpha hydroxy carboxylic acids” (AHAs) – e.g. citric acid, lactic acid, tartaric acid, mandelic acid, glycolic acid and isocitric acid - and their close structural relatives such as ketoglutaric acid, succinic acid, fumaric acid, maleic acid. In fact, the hydroxyl acids are known as being the most widely used “anti-ageing agents” [7]. Many of these small organic compounds are part of the metabolic or catabolic processes in living cells, for instance as part of the famous Krebs cycle. Exactly this fact, being a member of crucial metabolic pathways, explains the main “modes of action” of these acids. Many of them have PIC.: https://www.thoughtco.com/citric-acid-cycle-373397, by Evelyn Bailey

they not been used in cosmetics for years. It was shown that just have a superficial, more physico-chemical effect by lowering the pH and/or peeling off old skin cells. They actively involve in metabolic pathways. So, they contribute to reversing the effects of photoaging skin elasticity, tone and hydration. improving wrinkles, and As the metabolism of skin cells doesn’t differ substantially from that of other cells, these acids also should have a positive impact on other living tissues. There are many receptors for hydroxy- carboxylic acids and it they regulate homeostasis under changing metabolic and dietary conditions, by controlling metabolic, immune, and other body functions [8]. is known that In fact, prominent members of these acids have found extensive use with many aspects of life supporting, rejuvenation and curing diseases [9, 10]. So, inflammatory, Hydroxypropanoic analgesic anti- antimicrobial acid shows and activity and is used as an energy source by the brain when blood glucose level is low. Hydroxybutyric used neuroprotective therapeutic nutrient cataplexy and narcolepsy. acid is as a to treat fumarate (DMF) is known to be an Dimethyl effective novel oral the treatment of relapse-remitting multiple sclerosis (RRMS) [11] therapy used for treating Maleic acid is quite beneficial fibromyalgia and chronic fatigue syndrome and can have desirable effects on your oral health as well [12]. for Malic acid improves overall muscle performance, and this can ease chronic fatigue syndrome (CFS). The acid also boosts energy levels and improves the overall condition status. Malic acid is also used to boost sports performance when taken in supplement form [13]. STRUCTURES OF PROMINENT HYDROXYL ACIDS PIC.: structures of prominent hydroxyl acids by ChiroBlock

and inhibits rate of synthesis the citric acid cycle of Alpha-Ketoglutarate: Alpha-ketoglutarate (AKG) is a key molecule in the Krebs cycle determining the overall the organism. It is a nitrogen scavenger and a source of glutamate and glutamine that stimulates protein protein degradation in muscles. AKG as a precursor of glutamate and glutamine is a central metabolic fuel for cells of the gastrointestinal tract as well. AKG can decrease protein catabolism and increase protein synthesis to enhance bone tissue formation in the skeletal muscles and can be used in clinical applications. In addition to these health benefits, a recent study has shown that AKG can extend the lifespan of adult Caenorhabditis ATP synthase and TOR. AKG not only extends lifespan, but also delays age-related disease. The advances of AKG in the its physiological functions and applications were summarized in a study [14]. elegans by content of inhibiting by lowering oxidative Citric acid, in turn, shows effects on the brain and liver in lipopolysaccharide-treated mice. Thus in this model of systemic inflammation, citric acid (1–2g/kg) decreased brain lipid peroxidation and inflammation, DNA fragmentation [15]. damage, stress liver and In addition to many other beneficial effects on health, succinic acid was discovered to efficiently mitigate cerebellar mitochondrial OXPHOS dysfunction, neurodegeneration and ataxia in a mouse model [16]. Last but not least, isocitric acid (ICA), a hitherto underestimated hydroxy acid, has been found to exhibit quite a number of advantageous properties.

I soci tri c Aci d (I CA) – th e “h i dden g em” ICA is one of the last ubiquitous natural com- pounds - and the only intermediate of the Citric Acid Cycle – that is not easily accessible either via a biotechnological approach or by means of a chemical synthesis. This stands in striking contrast to the so far unmet need based on the unique properties and potential applica- tion fields of the com- pound. Just to mention some of the most favourable properties of ICA: • • • factor-alpha (TNF )’s it extends the life span of fly [17] wrinkles can be effectively treated [18] it lowers zinc induced neuro toxicology and prevents cerebrovascular dementia [19] it positively influences the coagulation properties of blood [20] it inhibits superoxide release and tumor necrosis release [21] ICA is the only metabolite able to unlock succinate dehydrogenase and therefore promote cell respiration, also during long and intense stress [22] The so called “cryptoporic acids” being sesquiterpenoidic ICA ethers, strongly inhibit the release of superoxide anion radicals and show anti-tumour promoting activity [23] ICA generally reduces oxidative stress [24] ICA glucocorticoid-induced cataract formation in eyes [25] ICA relieves the neurointoxication against the high concentration of lead and molybdenum salts, restores the memory and accelerates the learning [28] It improves the spatial memory [26] prevents • • • • • • • • PIC.: isocitrate structure by ChiroBlock To sum up all these positive properties: What else does it mean when one enjoys less oxi- dative stress, a better memory, eyes, more energy on a cellular level, less wrinkles and a longer life span? It means to remain young(er)! better ICA was shown to exhibit superior anticoagulation properties that could be used both in diagnostics and also in vivo with blood infusions and other biological fluids [27]. Based on its properties, it is reasonable to assu- me that ICA could replace all anticoagulants currently used in blood and plasma bags (mainly based on citric acid) and a great share of anticoa- gulants employed with the hundred of millions of diagnostic sampling tube kits doctors around the world use per year [27a]. So, why not to take ICA on a regular basis to im- prove our cells’ viability? Why not adding it to drinks, to functional food? Why not adding it to sophisticated cosmetic products? As a natural compound occurring in every living cell, there are definitely neither regulatory issues nor toxicity concerns that prevent industry from applying this substance? The answer is as simple as surprising: The exploi- tation of these market potentials so far was only prevented by the lacking availability of ICA. What is the problem of getting hold of this ubi- quitous “sister compound” of the citric acid that is manufactured on a million tons scale per year?

First, let’s have a look at the structure of ICA in comparison to that of citric acid: PIC.: structure ICA vs. citric acid by ChiroBlock Both look very similar: 3 carboxylic acid groups, one hydroxyl group and even the same number of carbon and hydrogen atoms resulting in the identical molar weight. However, a second look reveals the main difference: It’s all about geometry! ICA is not symmetric as citric acid and furthermore, it bears two stereogenic resulting in four! different (geometric) isomers: centres PIC.: 4 different geometric isomers of ICA by ChiroBlock of a forms Only one of these four isomers occurs in nature and is of real relevance for any application. It is quite obvious that preparing one of these four geometric “non-symmetric” compound is much more challenging than making the comparatively simply structured citric acid. Of course, there exist ways of either synthesizing ICA or extracting ICA from natural sources. However, none of these approaches have proved to be economically feasible so far.

The following scheme summarizes both the methods of ICA production and its (potential) applications: PIC.: methods of ICA production and its (potential) applications by ChiroBlock Method of Preparation Problems Natural material extraction [29] Chemical total synthesis [30] Lowest efficiency: 250 kg of leaves give only about 16 g, hardly scalable >6-8 steps, Yield < 20%; not scalable; mix of isomers Biotechnological Approaches [31] Yeast + organic substrate: mix of isocitric + citric acid (<70 : 30) + other carboxylic acids; separation hardly downstream possible processing); tendency of ICA to get decarboxylated racemized and/or dehydrated during work-up / formulation and/or (costly PIC.: methods of ICA preparation and their problems by ChiroBlock

All approaches face serious problems that result not only in a lacking availability of ICA (the pure isomer is only offered in tiny amounts resulting from an extraction process of leaves) but also in extremely high prices. The following table shows the prices and the annual production amounts of some of the small organic (hydroxyl) acids as average for the years 2017-2019: It is obvious, even for everyone not involved in this matter, that such prices as shown for ICA are prohibitive for any application – regardless how tempting or beneficial they may look. These prices simply reflect the efforts required to make / the consequence of patented processes or a general shortage of suitable starting materials. to isolate ICA – they are not PIC.: prices and the annual production amounts of some of the small organic (hydroxyl) acids as average for the years 2017-2019 by ChiroBlock CompoundMalicacidGlyoxylicacidSuccinicAcidAlpha-KetoglutaricacidCitricacidICA(racemate)ICA(purenaturalisomer)Ann.ProductionAmount[thsd.Tons]~250~4.5~30~1~2,500estm.:0.025estm.:0.001Priceperkg[$/kg]~14.00~40.00~20.00~63.00~1.30~18,000.00~240,000.00

Problems wi th bu lk sou rci n g of i soci trate - wh y i s i soci trate n ot as promi n en t as th e oth er compou n ds? What exactly are the problems that make this compound so expensive? The issues with extracting ICA from natural materials are quite obvious: While ICA is present in every living cell, it is only a minor component. So, the ratio citric acid : ICA in orange juice is about 150 : 1 (with about 40 mg CA / ml in high content material such as lemon juice). While this ratio can be used to prove the authenticity and origin of orange juice, it shows that it is no economically for manufacturing kilograms or even tons of this material. Chemical syntheses have to cope with other problems: creating the right isomer and to avoid approach feasible decomposition during conversion and work-up. Many chemical routes have been published – starting as early as 1889 [32] – but either, only a mixture of all isomers was formed, or – with stereospecific approaches [33] – the synthesis the work-up was extremely tedious and/or resulted or decomposition products (ref. PIC: synthesis with unwanted ICA derivatives or decomposition products) ICA-derivatives unwanted in One example of a chemical synthesis relying on “pre-formed” stereogenic centers is depicted below [33 c] (ref. PIC: synthesis relying on pre- formed stereogenic centers). PIC.: isocitrate synthesis with unwanted ICA derivatives or decomposition products by ChiroBlock PIC.: ICA synthesis relying on pre-formed stereogenic centers by ChiroBlock

While here, the malic ester already is „chiral“ and the danger of getting four isocitric acid isomers was banned, it is obvious that such a lengthy way using expensive and partially toxic reagents doesn’t open the door to a real availability of ICA. The starting material already costs nearly 2 $/g, the overall yield to the lactone 1 is only 15% on small scale (and hardly scalable), step f is feasible in principle, however requires special efforts and precautions to get the pure and dry product. of any The strengths of chemical approaches are the absence (related) hydroxyl carboxylic acid and the avoidance of water as solvent that makes a final isolation of the highly hydrophilic product difficult and costly. other The main drawback, however, of not being able to perform a really stereospecific reaction (avoiding any other isomer) outweighs these positive aspects. Biosynthetic approaches are another option and many procedures based on either fungi, yeasts or enzymes have been published so far [34, 35]. As all these processes are based on the natural bio- transformations in cells, they lack the main drawback of chemical conversions and only yield the natural enantiomer. However, also the “sister acid produced citric the main by-product of and compound” constitutes such syntheses. Together with related (hydroxyl) carboxylic acids these “impurities” need to be removed. This poses a problem as all these species are very similar and additionally they are extremely well soluble in water. Removing them from a typically watery matrix is difficult and costly. A number of alternative working-up procedures have been developed – including adsorption on charcoal, precipitation of salts of low solubility, chemical intermediates (mainly lactone 1) and electro dialysis. purification is via the selectivity of In summary, however, ICA versus CA on the one hand and the work-up efficiency on the other hand prevented an economically feasible production of ICA. So, is there any hope to find a cheaper way? The good news is: YES! found! It has already been Or better to say: Several ways have been paved during the last few months as a number of R&D institutions and companies around the world had intensified their efforts to finally solve this old problem.

I n n ovati ve/ n ew approach es to I soci trate – Maki n g th e compou n d avai lable on larg er scale So, now, two qualities of the natural form of ICI are available: • • the entirely pure isomer of ICI with only tiny amounts of CA as main “impurity” the pure isomer of ICI with no traces of CA but tiny residues of one of the other isomers as main “impurity” One example a Chinese company that managed to provide ICA by means of a newly developed enzymatic approach. Another example is the collaboration between two German research institutions (the Helmholtz Centre for Environmental Research - UFZ and the Institute of Non-classical Chemistry) and a chemical R&D company (ChiroBlock) supported by the Federal Ministry of Education and Research. The latter have developed two complementary new / improved routes towards ICA: a) b) a purely chemical approach and a biotechnological way substantially improved processing including a downstream PIC.: two complementary new / improved routes towards ICA by ChiroBlock While the very details of these processes are not yet available in the public domain, the following can be said about them: and/or that have to make sure that there is no biochemical conversion in the production history. The drawbacks of chemical and biotechnological approaches were successfully addressed: • • The chemical route starts with a cheap substrate, doesn’t require expensive or toxic reagents and yields the ICA free of CA with only one of the isomers as minor impurity. This way is of importance with any products and applications that are sensible towards traces of citric acid downstream The biochemical transformation, in turn, shows a high stereo selectivity (no traces ICA isomers) and a completely of improved processing without expensive and sophisticated purification operations. It would be the quality of choice for applications that tolerate traces of citric acid and water and that require a very low environmental impact.

This would not be commercially relevant if these synthesis processes could not be performed on large scales. In fact, they can! So, ICA, this wonderful natural compound full of potential with many (prospective) applications, now became available from a number of producers and vendors. Scientists managed to remove the bottlenecks in ICI production and opened the door for product developers in the pharma, and food industry to create new drugs, active ingredients and vitalizing agents for the benefit of all of us. cosmetics, diagnostics The prices of ICA depend on the different qualities and are still far from those of its cheap sister compound. However, they continue to decline with a rising demand and broader application fields. Links to ICA sources: Sigma Aldrich IndustryStock WLW Chem2market Pinpools Pinpools II Chemexper Chemexper II Chembuyers Guide Th e G reat fu tu re of i soci trate Now, with having several production processes and ICA sources available, the development of new final products based on the outstanding properties of ICA is going to get a boost. One doesn’t need to be prophetic to predict the first two main application areas: • • anticoagulants and activating, rejuvenating, energizing food and cosmetics And here scientists (modest) contribution to the Fountain of Youth becomes apparent: Providing the natural functional material every living cell needs to avoid negative stress, destructive metabolism and – hence – ageing! In other words: ICA products will be one essential factor helping to push the life span of humans beyond the 100 years without taking the hitherto unknown risks of the immature stem cell or gene editing technology. We are confident that ICA – as hidden gem, as hitherto ugly duckling – will now start to capture its place among (and above) the other (hydroxyl) carboxylic acids. Be part of the story and help to develop ICI based products!

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[27a] Isocitrate blood collection tubes are superior not only to citrate tubes but also with respect to EDTA and to NaF/heparin tubes. Such ICI buffers reduce in a gentle way the pH of the collected blood from 7.4 to about 6.5 and the concentration of free calcium ions from 1.3 mM up to 0.001 mM. At moderately reduced pH and calcium ion concentration, several blood analytes will become more stable than with prior art techniques. These analytes include among others coagulation factor V, coagulation factor VIII, homocystein, glucose, prostata specific antigen (PSA), soluble fibrin and complexes between protein C and its inhibitors. In addition there are cost savings for users because isocitrate will allow the use of fewer sample collection tubes, because not a number of different tubes have to be used, but the isocitrate tube is sufficient for most tests An isocitrate tube can be marketed as a universal blood collection tube and thus greatly impact on the collection tube market. Isocitrate plasma is attractive for the same reason as the isocitrate tubes, i.e. increased stability of several important clinical chemistry analytes. Blood bags and agents for cell preservation is an area in which isocitrate will find multiple applications. In present day blood banking, the blood and all blood products are anticoagulated by citrate. Isocitrate could here provide an advantageous alternative in that the concentration of free calcium ions is not reduced to the same extent. This facilitates the use of the blood products by reducing the adverse effects – bleedings, cramps and gastrointestinal discomfort – caused by the drop in free calcium ion levels induced by the citrate especially when larger quantities of citrated blood products are administered. This is especially true if liver functions are subnormal. Also the quality of infused blood products will be improved, i.e. the hemostatic value of infused plasma will be increased by reduced loss in activity of coagulation factor V and VIII which are destabilized at low levels of free calcium ions. Of greatest importance, however, is the fact that isocitrate plasma may be sufficiently stable to allow refrigerated storage (instead of storage at temperatures < - 16°C). [28] Morgunov, I.G.; Kamzolova, S.V.; Karpukhina, O.V.; Bokieva, S.V.; Inozemtsev, A.N. Biosynthesis of [29] [30] [31] [32] [33] [34] [35] isocitric acid in repeated-batch culture and testing of its stress-protective activity. Appl. Microbiol. Biotechnol. 2019, 103, 3549–3558. Vickery HB; MethodsEnzymol. 13 (1969) 601-609. a: Vickery HB; Science (Washington, DC, United States) 132 (1960) 892; b: US3318944; c: US4123458; d: US4282156 Greenstein et al.; J. Am. Chem. Soc. 77 (1955) 707-712 a: Heretsch, P.; Thomas, F.; Aurich, A.; Krautscheid, H.; Sicker, D.; Giannis, A. Syntheses with a Chiral Building Block from the Citric Acid Cycle:(2R, 3S)-Isocitric Acid by Fermentation of Sunflower Oil. Angew. Chem. Int. Ed. 2008, 47, 1958–1960; b: Kamzolova, S.V.; Dedyukhina, E.G.; Samoilenko, V.A.; Lunina, J.N.; Puntus, I.F.; Allayarov, R.L.; Chiglintseva, M.N.; Mironov, A.A.; Morgunov, I.G. Isocitric acid production from rapeseed oil by Yarrowia lipolytica yeast. Appl. Microbiol. Biotechnol. 2013, 97, 9133–9144; c: Effects of Medium Components on Isocitric Acid Production by Yarrowia lipolytica Yeast, November 2020, Fermentation 6(4):112 Fittig, Miller; Justus Liebigs Annalen der Chemie 255 (1889) 43-46 a: Seebach D, et al; Org. Synth. 63 (1985) 109; b: Schmitz C, et al; J. Org. Chem. 61(5) (1996) 1817. Friedrich-Freksa; Martius; Zeitschrift fuer Naturforschung; vol. 6b; (1951); p. 2; Krebs; Holzach; Biochem J 52(1952); 527- 528 a: Kamzolova SV, et al; Applied BiochemMicrobiology 51 (2015) 249–254.; b: Aurich A, Hofmann J, Oltrogge R, Wecks M, Gläser R, Blömer L, Mauersberger S, Müller RA, Sicker D, Giannis A; Org. Process Res. Dev. 21 (2017) 866−870. c: Holz M, et al ; Appl. Microbiol. Biotechnol. 81 (2009) 1087-1096.; d: Laptev IA, et al ; Appl. Biochem. Microbiol. 52 (2016) 699-704.

Pictures: All pictures without subtitle by Adobe Stock This whitepaper is part of a research project conducted by The Helmholtz Centre for Environmental Research, the Institut für Nichtklassische Chemie e.V. (INC) and ChiroBlock sponsored by the Federal Ministry of Education and Research.