I. Isotopes in the Cell?

[first posted: 1.24.17/most recent edits & additions: 2.28.17]

Preamble: this conjecture about the problematic role certain combinations of isotopes may play when they are woven into the DNA molecule in proximity to each other is a prelude to the more specific discussion about mutations found in Topic VIII: Alternative Structures to Watson-Crick in Bound Chromosomal DNA? Conjecture about the role of isotopes in determining the fine-scale sense of chirality is discussed in Topic VII: Isotopes, Nuclear Spin & Chirality. The disruptive role isotopes have on the precession rate of rotating elements in a gravitational field is taken up in Topic VI: Molecular Precession, Gravitation, and Circadian Rhythms.

Hypothesis: The composition of the cytosol is constantly undergoing change due to the flux of molecules passing through the cell membrane between the cytoplasm and the inner cell. While the large scale functional properties of these molecules is determined by their chemical composition, a finer-scale distinction can be found in their isotopic composition. Isotopes (natural ratio shown in parentheses) such as 15N (0.3%), 13C (1.1%), 17O (0.04%), 18O (0.2%), or 2H (0.01%), are also ingested as the cell grows and undergoes mitosis. Heavy isotopes slow down the chemical reactions of their host molecule, dampen their oscillation frequency, and alter their function. As the cell ages, in an effort to better maintain cell performance, a re-distribution of isotopes may occur - preferential shuffling heavy isotopes to less critical areas of the cell. This can lead to an increased likelihood of isotopic clumping further downgrading cell performance. The basis for this conjecture is outlined below and two experimental programs are outlined below that might test this hypothesis.

Why isotopes? And, in particular, why heavy isotopes? Heavy isotopes also have surplus neutrons, and whenever a heavier isotope is substituted for a lighter isotope, the vibrational frequency of that molecule slows down, and the rate at which chemical reactions occur involving that molecule is also slowed - this is known as the kinetic isotope effect*. Heavy isotopes form shorter bonds than and when they join with other atoms, the resulting molecule will have a slightly different geometry and a different zero-point energy. Thermodynamic stability is, therefore, related to the isotopic composition of the molecule. It has also been found that heavy isotopes have a tendency to clump together. Thus we must consider the possibility that over the period of a human lifetime, such clumped isotopes might accumulate within pockets of the cell to form extended regions where the mean vibrational energy is significantly dampened - or significantly altered from the rate at which energy was previously exchanged when the cell was young.

For practical reasons, we most often treat molecules as being comprised of their most common isotopic forms (C12, N14, O16, etc..), but, of course, every carbon atom, every nitrogen atom, and every oxygen, hydrogen, and phosphorus atom – up and down the periodic chart - come in both their most common forms and either heavier or lighter forms. The disruption in rhythm caused by isotopes is proportional to the relative change in mass, thus the isotope effect of is greater significance in light atoms such as H, Li, B, C, N, O, than in heavy atoms. Altering the rhythm of core molecules such as those found in gene sequences within DNA can, therefore, threaten their proper function. How the cell manages molecules that host isotopes with abnormal rhythm becomes the important focus of this section.
* (For an introduction to the Kinetic Isotope Effect, see: https://en.wikipedia.org/wiki/Kinetic_isotope_effect. For an article on clumped isotopes, see: http://www.carbonateresearch.com/clumped_isotope and for information about first and second order spectrum shifts caused by isotopes, see: http://chem.ch.huji.ac.il/nmr/techniques/other/isotope/isotope.html)

In biological environments like the cell, where there are roughly 10^12 molecules - we must expect to find a full spectrum of isotopes. Altering the neutron count by one in heavy atoms such as Zinc (atomic mass ~ 65 amu) or Bromine (atomic mass ~ 80 amu) doesn't change their inertial properties much. But, adding an extra neutron to a small atom like Carbon (atomic mass ~ 12 amu) and its atomic mass is increased by about 8%. And, even more drastically, if a neutron is added to hydrogen, it produces deuterium, which doubles its atomic mass. This is important because, when two atoms or molecules become coupled, it is their respective atomic mass that is fundamental to their oscillation frequency. That frequency determines how the coupled structure interacts with its environment - either dampening or exciting vibrations in neighboring molecules. Thus, we can expect that when off-rhythm isotopes get bound into amino acids, proteins, and every other group of molecules - including nucleotides - they may alter the functional characteristics of those molecules. Schematically, an eukaryotic cell - dotted with isotopes - can be expected to look much like the image below:

Of course, as noted above, isotopes are found everywhere in nature. With every gulp of air we breathe, with every sip of water we drink (stomach), we ingest isotopes. Thereafter, with each beat of the heart, these isotopes are pumped through our body (breast, uterus, liver, prostate…) until they get expelled from the body (bladder, colon, skin…) back into the environment where they pose no more threat to our delicate systems. The ratio of heavy or light isotopes is typically small - but it is much higher than the 10^-20 threshold discussed in our working hypothesis for possible links to cancer. Throughout the lifetime of a cell, statistically, many combinations of potentially hazardous isotopes will find their way into proximity to each other. Thus, it is not difficult to imagine how - if the cell does not manage these isotopes properly - these off-rhythm combinations might cause problems. (For a discussion on how the biological molecules in contained fluids establish their local definition 'up' and 'down', and thus which direction they tend to fall, see Topic V: Protein Shape and Terminal Velocity in the Cytosol?)

Clumped Isotopes: Of relevance here are developments in the fields of geology and planetary sciences where it has been found that naturally occurring heavy isotopes tend to bond or clump with other heavy isotopes with a temperature dependence. Accordingly, the ratio of certain combinations of clumped isotopes can be used for paleoclimate reconstruction. From the journal of Earth and Planetary Science Letters in 2007 (Volume 262, Issues 3–4, 30 October 2007, Pages 309–327 http://dx.doi.org/10.1016/j.epsl.2007.08.020); in an article by John M. Eiler (Division of Geological and Planetary Sciences, California Institute of Technology):

"Clumped isotope geochemistry is concerned with the state of ordering of rare isotopes in natural materials. That is, it examines the extent to which rare isotopes (D, 13C, 15N, 18O, etc.) bond with or near each other rather than with the sea of light isotopes in which they swim. Abundances of isotopic ‘clumps’ in natural materials are influenced by a wide variety of factors. In most cases, their concentrations approach (within ca. 1%, relative) the amount expected for a random distribution of isotopes. Deviations from this stochastic distribution result from: enhanced thermodynamic stability of heavy-isotope ‘clumps’; slower kinetics of reactions requiring the breakage of bonds between heavy isotopes; the mass dependence of diffusive and thermo-gravitational fractionations; mixing between components that differ from one another in bulk isotopic composition; biochemical and photochemical fractionations that may reflect combinations of these simpler physical mechanisms; and, in some cases, other processes we do not yet understand. Although clumped isotope geochemistry is a young field, several seemingly promising applications have already emerged."

And, from Harvard University:
(see: http://environment.harvard.edu/clumpedisotopes)

"The study of multiply-substituted isotopologues (or clumped isotopes) is a growing field within Earth sciences and geochemistry.  To date, most research has focused on either clumping in carbon dioxide or carbonate minerals with applications to atmospheric budgets, paleothermometry and diagenesis. As we meet for the 3rd International Workshop on Clumped Isotopes we welcome back the existing carbonate and CO2 clumped isotope community and welcome newcomers studying clumping in O2, organic molecules and other systems."

And from the journal Science;
(24 Apr 2015: Vol. 348, Issue 6233, pp. 431-434 DOI: 10.1126/science.aaa6284)

"What controls clumped isotopes?
Stable isotopes of a molecule can clump together in several combinations, depending on their mass. Even for simple molecules such as O2, which can contain 16O, 17O, and 18O in various combinations, clumped isotopes can potentially reveal the temperatures at which molecules form. Away from equilibrium, however, the pattern of clumped isotopes may reflect a complex array of processes. Using high-resolution gas-phase mass spectrometry, Yeung et al. found that biological factors influence the clumped isotope signature of oxygen produced during photosynthesis
..."

While the above referenced research focuses on the geological implications of clumped isotopes, there is reason to consider what affect such rare combinations of clumped isotopes might have within the biological environment of the cell. This becomes the basis for a discussion that continues in Topic VIII: Alternative Structures to Watson-Crick in Bound Chromosomal DNA?

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At normal body temperature, the molecules in our cells collide roughly 10 billion times per second. Activity at the molecular level might be compared to a crowd of musicians each tuning up different instruments. If - to extend the analogy - from within the crowd, two musicians start to harmonize, and then two more join those first two, and then two more after that, then slowly, from within the cacophony that previously existed, a rhythm emerges. And, if all the participants join in, then - just perhaps - a symphony is heard. Such is a fair model for the cooperative molecular behavior in the cell when it is functioning properly. However, when heavy or light isotopes are substituted for their more common atomic forms, then this may be seen as analogous to switching musicians and their instruments in that symphony. What happens to the music, if, say, a musician playing a French horn gets tired and a trombone player must sit in for them? Maybe, if the number of musicians is large, the difference goes unnoticed. But, if, over time, many such substitutions are made - oboes for flutes, cellos for violins, and tubas for trombones, how would we expect the music to sound?

A few more less-than-scientific-but-still-useful analogies are worth relating here as they pertain to strategies the cell may have adopted to help keep isotopes and their abnormal rhythms out of the molecules linked to, or embedded within, DNA.

In Berkeley, there is a well-known used bookstore on Telegraph Avenue called Moe's. The owner, Moe, was notorious for his resistance to buying back books that were not of keen interest to the community of well-educated book buyers. In Moe's words: “Any bookstore that buys back bad or even mediocre books will end up with a book store filled with books no one wants. That is business suicide.” By analogy: "Show me a cell that ends up with a shelf full of isotopic nucleotides to choose from when building its DNA or mRNA and I will show you a cell that the local biological community does not want." (Heavy) Isotopic encrusted nucleotides will react more slowly, thus failing to operate as efficiently as their fellow isotope-free nucleotides, and will thereafter contribute to the degradation of the cell.

If someone who goes to the market to buy apples for a pie that they have to bake that night - and thus, it is not an option to not buy apples - then they will have to choose from whatever the market offers. If the crop is poor, some apples that might have been rejected on another occasion, will now have to be selected.

To be more explicit about how these analogies relate to cell biology; whenever DNA is damaged, such as the 1 million lesions that are hypothesized to occur in DNA each day, or whenever DNA duplicates itself during S-phase, the nucleotides and molecules used for these processes must be selected from whatever is available within the local environment - even if these nucleotides and molecules are dotted with isotopes. And, thus - while cancer is known to be caused by mutations in existing genetic material - might cancer also be thought of as a result of sustained construction using materials which are statistically the ‘best' choice at the time, but which are selected from a pool of progressively deteriorating choices which already possess a tendency for mutation? As we age, don't all of our respective biological pools include an increasing percentage of ‘lesser choices’. Might a cancerous cell be steered down a road where it can’t correct the imbalance in its internal rhythm induced by isotopes fast enough? In response, might the cell try to repeatedly grow out of its predicament...but when the pool is not as pure as it once was, and contains a higher ratio of off-rhythm isotopes, then won't each new generation of cells become a little more troubled than the previous generation? (tumor)?

It may justifiably be argued that DNA has many repair mechanisms that can locate and remove such misbehaving nucleotides. If there were only a few isotopes that were throwing the internal rhythm of the cell off, then perhaps these repair mechanisms could sufficiently purge the nucleus of the potentially hazardous combination of isotopes conjectured about in this section. But if, as the cell ages the ratio of isotopes increase, then it can also be argued that during the repair process - when our tightly wound double helix is opened up and exposed to whatever isotopes might be lurking nearby - there is also an increased chance that wrong isotope-dotted nucleotide will find its way into one of the DNA strands and woven into the molecule upon closure. By such conjecture, if nucleotides are (metaphorically) grabbed from a storeroom filled with aging isotopic nucleotides, the repair process might - at least in some instances - exacerbate the problem and give us a cell that no one wants (Moe’s theorem).

Two experimental programs are outlined below to test this conjecture... but first, let us extend the discussion to make it more specific to the geometry of DNA.

Relevant to DNA and the H-bonded N-H---O (or N-H---N) core of molecules that critically hold opposite DNA strands together, we can ask: what are the odds that we find the less common isotopic combination of atoms depicted below rather than the most common isotopic form, 14N-1H-16O?

Well, that would require a role of the dice where we land 15N instead of 14N (odds: 1/282), and 2H instead of 1H (odds: 1/8695), and 18O instead of 16O (odds: 1/487). A statistically rare event with a ‘one in a billion’ chance of occurring. First impression: not very likely. But on second look, we remember that we’ve got 3 billion base pairs in our chromosomes – we expect to find three such ‘one in a billion’ type combinations in every cell. And, we’ve got 10^13 cells rolling the dice all day long! It cannot be overemphasized how very likely 'unlikely events' are when modeling biological events that include moles of particles (10^23).

Relevant discussions about the possible problematic role isotopes play can be found in each of the home page topics. This discussion will continue more in depth in Topic VIII: Alternative Structures to Watson-Crick in Bound Chromosomal DNA?

Extended Conjecture that Relates to Topic III: Gravitational Cycles & Induced Torque Into the Cell?
Gravitational potential energy is a much finer currency for energy than the energy released when ADP is converted into ATP. An interesting coincedence may be noted here: the small (delta E)=(mgh) energy change that results when lifting a molecule of ADP from one side of the earth to the other relative to the sun’s gravitational field turns out to be of similar order as the energy change in ADP to ATP.  Might the complicated sequence of acceleration patterns that convert ADP into ATP in the cell somehow collapse or ‘harvest’ that potential energy into the resulting chemical energy associated with the change in bond structure?  And if so, might the conduit for these energy changes be found in the nuclear spin states of the participating molecules?

Below are two experimental projects related to isotopes that we believe would be valuable to be performed.

Exp. 1: Mapping isotopic concentration in the body.
It would be valuable to have a detailed map of the general distribution of isotopes in the body. This will be an extensive task for we need to first establish a background density for a full spectrum of isotopes {H, C, N, O, P…} taken from different tissues through the body {lung, heart, breast, prostate…and stem cells} from males and females from different age groups. A natural ratio of isotopes {2H/1H ~ 1/6000,13C/12C...etc} is expected, but of interest would be the statistically significant deviations from the expected values. Variations need to be broken down into at least five categories of cells: normal youth cells, normal adult cells, cancerous youth cells and cancerous adult cells.

 

Exp. 2: Mapping isotopic concentration within the cell.
If Exp. 1 reveals a variation from the natural isotopic spectrum, then it would be valuable to pursue a more specific breakdown of isotopic distribution and composition within the cell itself.  That is, we need the isotopic ratios for molecules pulled from DNA, the nucleolus, the cytosol, ribosomes, the cytoplasm, etc...  Again, we need a level of accuracy that will reveal any statistically significant results.

A good introduction to the distribution of isotopes in the body can be found at:
[http://hps.org/publicinformation/ate/faqs/faqradbods.html]

One exemplary quote about potassium isotopes is provided here:

"The amount of the radioactive isotope (potassium 40K) in a 70-kg person is about 5,000 Bq, which represents 5,000 atoms undergoing radioactive decay each second. Second, 40K emits gamma rays in a little over 10 percent of its decays and most of these gamma rays escape the body. A gamma ray is emitted in about one out of every 10 disintegrations of 40K, implying that about 500 gamma rays are produced each second."

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For reference: below is a graphic that illustrates the likely abundance ratio of specific N-H---O isotopic configurations and their expected count per cell. A more detailed chart can be found in Topic VIII: Alternative Structures to Watson-Crick in Bound DNA?


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