Discovered nearly a century ago [1], there are many roles of adenosine triphosphate (ATP) in living organisms from then and to the present; the principal role or primary function is energy metabolism. The mitochondrion is the main source for the production of ATP in eukaryotes, whereas in arachaea and prokaryotes, having no mitochondria, the source for these realms is the proton pump. Advances in phosphorus-31 nuclear magnetic resonance (³¹P NMR) enabled the first report of excessively high millimolar (mM) concentrations of ATP in the mitochondrial laden, living intact skeletal muscle tissue, a finding that was not unexpected [2]. The high mM concentration of ATP was anticipated because of the high energy demand of skeletal muscle. However, soon thereafter, a similar excessive amount of ATP was unexpectedly discovered in the intact living crystalline lens [3]. This finding presented a conundrum, because the lens has a paucity of mitochondria and served a function that was metabolically quiescent. In fact, the lens undergoes a process of denucleation with concomitant loss of intracellular organelles, including mitochondria, during maturation [4], and thus would have a declining concentration of ATP with aging [5], as we expected.

Paradoxically, archaeotic and prokaryotic microorganisms, having no mitochondria, also have excessively high millimolar concentrations of ATP [6]. This observation presented an even greater enigma, since microorganisms, being completely self-contained, are metabolically known to have a high energy demand. More perplexingly, the concentration of ATP needed for metabolic function requires only a small portion of the high concentration of ATP reported [6, 7]. Considering the Michaelis constant, only micromolar (µM) values are required for ATP-driven cellular processes [8]. Biochemical analysis reveals that only a small portion of the high cellular concentration of ATP is required for all the known functions of ATP, combined [6], thus, why do both microorganisms and intracellular lens tissue have ATP at the level of an order-of-magnitude greater than is necessary. This becomes especially important considering that nature is ordinarily conservative in the generation of high-energy phosphatic metabolites, such as ATP. When the ATP concentration is examined among different cells, tissues, and organs and even across the phylogenetic tree, we discovered that ATP ordinarily exists in exceedingly high concentrations [6].

Both microorganismal cells with no mitochondria and lenticular fiber cells, many of which have no mitochondria, most notably in the nuclear region of the lens [9], carry out glycolysis with phosphorylated intermediates, turn on and off protein synthesis and nucleic acid functions and the other myriads of functions requiring ATP [7]. A potentially major role of ATP, however, has been completely overlooked, and may serve a role of paramount importance: ATP is a hydrotrope [10, 11, 12]. The hydrotropic function of ATP has been postulated using both cellular and tissue homogenates [11] and hypothesized using the living functioning lens organ [12]. Without the hydrotropic function of ATP, there can be a decline or loss of protein solubility and consequent protein aggregation, which can include dysfunction or cessation of function(s) of enzymatic and structural proteins. Absence of hydrotropic ATP function can be detrimental to cellular, tissue, and organismal function and homeostasis [11]. Our more recent study revealed the high concentration of ATP exists across cells and tissues, species, and all three phylogenetic domains (eukaryota, archaea, and prokaryota) at the level of an order-of-magnitude higher than expected [6], but that is consistent with an intracellular role of ATP’s hydrotropic properties.

Evidence for such a role is as follows: Patel et al. [11] postulated that ATP functioned as a hydrotrope preventing protein aggregation in cellular and tissue homogenates. Greiner and Glonek [12] hypothesized that ATP prevented protein aggregation in the living intact crystalline lens organ demonstrating non-invasively the hydrotropic functional relationship between ATP and water and formulated a molecular model for this function. Protein aggregation in the lens is known to cause cataract [13], a disease which can occur in every human being that eventually causes impaired vision. Protein aggregation is believed to be the underlying pathogenic mechanism in the three most common age-related vision-threatening ocular diseases that can affect everyone with aging [13]. These diseases include age-related cataract, presbyopia and age-related macular degeneration.

Why does the high mM concentrations of ATP (ave. 4.4 mM) found in all Domains of the tree-of-life [6], exceed the metabolic needs of cellular metabolism by an order-of-magnitude? There is no significant difference between the concentration of muscle ATP and lens ATP even though these two tissues are at opposite ends of the metabolic spectrum [6]. Since cells and tissues with mitochondria and those without mitochondria present no significant differences in their ATP concentrations, this suggests that (1) the production of ATP may be more or less dependent on ATP’s role in cellular metabolism, which has been presumed to be the powerhouse or energy function of the cell, and (2) the cross-domain conservation of such a presumptive role for ATP among eukaryotic, archaeotic, and prokaryotic cells may represent a highly-conserved and fundamental concept underlying cell, tissue, and organ viability [6]. This does not mean ATP is not involved in energy metabolism or any of the other functions already attributed to ATP. ATP is a multifaceted molecule having many roles of variable importance [12]. Based on the above, ATP is likely principally functioning as a hydrotrope for which concentrations in the mM cellular and tissue homogenates range are required. This is logical, and along with the data presented previously by Patel et al. [11] in cellular and tissue homogenates and Greiner and Glonek [6] in a whole living organ makes this possibility compelling.

In all phylogenetic domains, reports have established that the excessively high mM concentration of ATP are present in studies of eukaryotic cellular and tissue homogenates [11], living tissues [14], and a living organ [6] as well as prokaryotes [15]. We question as to whether such highly-conserved concentrations may be present historically earlier in the more primitive microorganisms, the bacteria. Also, by extrapolation, since it appears that ATP is involved in prevention of protein aggregation and maintenance of protein solubility [6, 11, 12], we speculate that ATP functions in preventing protein aggregation in bacteria. Such function in the more primitive organism remains to be shown.

Considering the general belief that (1) mitochondria present in eukaryotes are derived from bacteria, and (2) since most of the ATP is generated from intracellular mitochondria, it may be unexpected that the ATP concentration also is excessive in archaea and prokaryotes. Our discovery, reporting high concentrations of ATP in a living functioning tissue, the lens [3], with its paucity of mitochondria relative to muscle was surprising as is the observation where there is scarce, or no mitochondria and the concentration of ATP is also high in archaea and prokaryotes [6]. This observation supports the concept that the ATP concentration is a highly conserved feature underlying cell, tissue, and organ viability. Whereas, the fact that the mitochondrial ATP supports the cellular engines of eukaryotic cells and the speculation that bacteria may be the organism from which the mitochondria were derived, it is clear that the prokaryote ought also to have a high concentration of ATP [6].

Concordant with the more highly specialized eukaryotic cellular tissues, single-cell eukaryotes and the procaryotic microorganisms ought similarly to have high concentrations of ATP [6]. Moreover, some microorganisms belonging to the archaeotic domain are known to have primordial roots [16, 17, 18]. Heritage of these microorganisms extends at least 2.5 billion years. As such, evolutionary heredity cannot be overlooked [6]. The initial function of ATP may have been as a hydrotrope predating ATP’s use as intracellular energy currency [6], since even in the absence of intracellular organelles e.g., a nucleus or mitochondria, the maintenance of microorganism function producing ATP at high concentrations indicates that millimolarity of ATP may have been a feature of the first functional progenotes [6].

The value of advances in the ³¹P NMR methodology offered, for the first time, ATP measurement in an intact living tissue [2]. Weaknesses in our hypotheses include: the NMR magnet field strength that improves signal detection with increased signal strength, the concentration of phosphorus-31 (the only naturally occurring isotope of phosphorus in living organisms) with a concentration much lower than protons as measured in ¹H NMR imaging, and the limitation on the number of living actively metabolizing species samples with known concentrations of ATP. Strengths of our hypotheses include the fact that the ATP measurements detected are unencumbered by degradation in sample preparation, since cells, tissues or organs are examined intact and living using ³¹P NMR technology.

Our findings of high millimolar ATP is a fundamental biological feature of a living cell. Our hypothesis predicts (1) ATP serves a critical molecular function which is foundational across all taxonomic domains for organismal homeostasis, and (2) the hydrotropic property of ATP prevents pathological protein aggregation and maintains protein solubility and, thus the health of cell, tissue and organ systems.

Conceptualization, JVG and TG; writing original draft, JVG and TG; Writing—review and editing, JVG and TG. Both Authors have read and agreed to the published version of the manuscript. Both authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

The authors thank Paula J. Oliver for her organizational support and editorial contributions and acknowledge Dr. Louis Collins for her commitment as a library scientist in the development of this work.

The Valarie and Walter Winchester Grant #533181, Schepens Eye Research Institute of Massachusetts Eye & Ear, Harvard Medical School, Boston, MA.

The authors declare no conflict of interest.