Aspects of Diagnostic Value of Hair Spectrometry for Content of Electrogenic Metals (Na, K, Ca)

After years of research, the authors have arrived at the conclusion that it is unjustified to use spectrometric analysis of Na, K, and Ca content in hair for integral assessment of the said metals’ “adequacy” in the whole body. This is evidenced not only by significant scatter of metal concentration values in the given substrate (hair) (by several orders of magnitude!) in practically healthy individuals but also by the specifics of Metal-Ligand Homeostasis (MLH) in the epidermis whose regulation differs fundamentally from systemic MLH control in the body in toto. Therefore, to avoid unjustified extrapolation to the whole body, any data of hair spectrometric analysis should be referred to this bio substrate only.

The reason for writing this article (which could be placed under the heading “Letter to the editor” or “Mini-review” in a scientific magazine) was the authors’ intent to point out the widespread fallacy among bioclimatologists that hair spectrometry for Na, K and Ca content can provide objective assessment of homeostasis of the said metals in the whole body. The idea of using hair as bio substrate for spectrometry in the study of MLG is not new (dating back several decades) and the spectrometric analysis methods in modern interpretation: Atomic Absorption Spectrometry (AAS), Plasma Atomic Emission Spectrometry (ICP-AES), Plasma Mass Spectrometry (ICP-MS) and others, being high-tech, are becoming increasingly popular over recent years. In Moscow alone, more than a dozen medical centers have appeared where the elemental status of the whole body is judged by the results of hair spectrometry. But how justified is such generalization? Despite the abundance of numerous publications on this topic in modern literature, there is not a single report where the validity of such an extrapolation would be proven. At the same time, the a priori, not supported by any facts, conviction that shifts in the MLH of the epidermis (hair) correspond to those in the whole body is taking on the characteristics of some sort of creed which is contrary to scientific analysis. That is why it seems relevant to initiate public discussion on this matter. What is the quantitative assessment of Na, K and Ca level obtained on a modern spectrometer? What is behind these numbers? To answer these questions, we need to recall some features of metal homeostasis. The human body (regardless of gender) contains ~3000mmol of sodium. 70% of this amount is the exchange pool, 30% is in the bone tissue [1]. Extracellular fluid (normally) contains 135-145mmol/l of sodium, while in cells it is only 4-10mmol/l.

Being the “main osmotic ions” of the extracellular space, where Na+ makes up 90% of all other ions, Na+ is directly involved in the regulation of water and electrolyte balance. This is facilitated by the high sensitivity of hypothalamic osmoreceptors which can be judged by a rather narrow range of fluctuations in the normal level of Na in plasma (135-145mmol/l), as well as by a change in the volume of extracellular fluid in response to sodium excretion within only 1%. It is known that 85% of sodium is excreted in urine (with a balanced Na diet), and only 15% is excreted through the skin (sweat glands). The content of K⁺ (the main intracellular cation) in the body of an adult weighing 70 kg is ~3500mmol. And although 90% of potassium is in free form and only 10% in bound form (erythrocytes, brain, bone tissue), the actual size of the exchange pool, due to the predominantly intracellular localization of K+, is rather modest. In the extracellular space, there is only 2% of the total amount of potassium (50-60mmol). The concentration of K⁺ in the cytosol is ~110mmol/l, in the extracellular fluid ~4mmol/l. However, it should be noted that the total volume of water in the extracellular space is half that in the cytosol (14 l vs 28 l resp.).

Permissible limits of normal fluctuations in the level of K in plasma are 3.6mmol/l to 6.3mmol/l. Even a slight violation of these limits (for example, decrease below 3.0mmol/l) can cause serious disturbances in cardiac activity. The polar difference in the predominant localization of the Na+ and K+ cations relative to the cell membrane is combined with the opposite direction of their electrochemical gradients: for Na+, it is inside the cell, and for K+, it is outward. The separation of cations relative to the cell membrane and the maintenance of this “inequality” becomes possible due to the constant work of the ATP-dependent membrane pump Na+/K+- ATPase, which pumps K⁺. ions into the cell and removes Na+ ions (in a ratio of 2:3 resp.). The resulting potential difference on opposite surfaces of the membrane (membrane potential) makes it possible to consider not only Na⁺./K⁺.-ATPase, but also Na+ and K⁺. cations as electrogenic. Calcium cations (Ca²⁺) are also among the electrogenic ones, the content of which in the human body is noticeably higher than that of other metals (25000mmol or 1000g per 70kg of body weight). 99% of Ca is in the bones of the skeleton and only 22.5mmol is in the extracellular fluid.

There is very little of this metal inside the cell-only 50nmol/lseveral orders of magnitude (!) less than in plasma or interstitial fluid, where [Ca²⁺] ~2.5mmol/l. The impressive “difference” of the electrochemical gradient that exists in Ca²⁺, which is necessary for the generation of an action potential, is mainly provided by the actively working membrane Ca²⁺-ATPase due to the energy of ATP molecules. Normally, permissible fluctuations in the plasma level of Ca are 2.2-2.7mmol/l. Let us now turn to the quantitative assessment of the level of Na, K and Ca, which gives us hair spectrometry (in mcg/g). How should the spectrometer readings be interpreted? The universality of the basic laws of cellular life justifies the following interpretation of spectrometric data:
i. for sodium, this is the amount of metal that is located mainly outside the epidermal cell (interstitial fluid), where [Na⁺] is ~10 times higher than [Na⁺] in the cytosol.
ii. for potassium, this is mainly an intracellular pool, because [K⁺] in the cell ~30 times higher than extracellular [K⁺].
iii. [Ca²⁺] in the cell is so small (50nmol/l) that the level of calcium in the hair, which the spectrometer shows, reflects the size of the extracellular pool of this metal.

At the Center for Biotic Medicine (Moscow), measurements of sodium (Na), potassium (K), and calcium (Ca) levels in hair were made using inductively coupled plasma mass spectrometry (ICPMS) on a NexION 300D spectrometer (Perkin Elmer Inc., Shelton, CT, USA). Under observation were practically healthy residents of Moscow aged 20 to 49 years (n=9991). Among them, 4999 (50.04%) men and 4992 (49.96%) women [2]. It should be emphasized that all these measurements were carried out in practically healthy individuals with no pathological symptoms. At the same time, the spread of individual values of metals was impressive: for Na, from 0.645mcg/g to 9240mcg/g; for K, from 0.045mcg/g to 6505.1mcg/g; for Ca, from 15.5mcg/g to 19338.9mcg/g. How can one explain such “tolerance” (insensitivity) of epidermal cells to pronounced (several orders of magnitude!) quantitative shifts of Na, K, and Ca, occurring mainly in the interstitial space of the epidermis? Note that much more modest fluctuations in the plasma level of the same metals are fraught with serious (and sometimes fatal) “failures” in the work of the most important functional systems of the body. This may be due to the absence in the epidermis and its appendages (as well as in other tissues that do not have electrical excitability) of peculiar “alarm receptors” (like hypothalamic osmoreceptors), which would be sensitive to shifts in MLH.

It cannot be ruled out that the epidermis, through the entire evolution of humans as a species, simply did not need such signal structures. What, then, can connect hair, as an appendage of the epidermis, with the homeostasis of metals in the body in toto, if we bear in mind that the skin does not play the main role in the processes of intake and loss of metals. 85% of sodium, for example, as already mentioned, is excreted by the kidneys and only 15% through the skin. Why, then, the kidneys and epithelium of the digestive tract, which are directly involved in the implementation of metal homeostasis, cause less confidence in the search for reliable MLH discriminators than hair? Unfortunately, it is not yet possible to answer this and other questions. It is also unclear whether there is any homeostatic control of the transmembrane transport of electrogenic metals at the level of the epidermis? The results of our research over recent years are inclining us towards an affirmative answer to this question. Evidence was obtained that homeostasis of K, Na, Ca in the derivative of the epidermis (hair) belongs to the phenomena of Self-Organized Criticality (SC) [3,4]. Among them:
a) a reliable linear relationship (Pearson) between the concentration values of Na and K in hair (r K-Na = 0.8-0.9, considering the [Na]/[K]-ratio), indicating the synchronous (criticality) work of membrane Na+/K+-ATPase [2].
b) the presence of a power-law relationship, confirmed graphically on a double logarithmic scale, between the content of metals in the epidermis (hair) and the number of individuals in certain intervals of numerical values for Na, K, and Ca, as well as the fractal nature of the distribution of the results of spectrometry of these metals [3].
c) conjugated (cluster) nature of quantitative changes in MLH against the background of oxidative/nitrative stress [5].

It should be added that the magnitude of the measured indicators of electrogenic metals in hair is related to the gender and age of the subjects. Significant gender-related differences in normal values were found in Ca (the “female norm” for Ca may exceed the “male” value by more than two times), and significant age-related differences were found in the content of Na and K in healthy individuals [3]. The concept of self-organized criticality helps to present the pool of Na⁺, K⁺-ATPases as a system of oscillators capable (when a sufficient density of active molecules on the cell membrane is reached under the influence of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) to “adjust” (synchronize) their own oscillations, thus passing into a critical (synchronized) state. Thus, ROS and RNS are activators of Na⁺, K⁺-ATPase. The actual activation of Na⁺, K⁺-ATPase occurs upon modification of the cysteine SH-groups in the protein molecule of this enzyme with the participation of ROS (oxidation) and/or RNS (nitrosylation). As a result, not only the number of actively working transporters (ATPases) increases, but also the size of the pools of transported ions (Na⁺ and K⁺).

These processes occurring at the level of the epidermis can play the role of local control of MLH for Na, K and Ca and not be dependent on the central regulation of ion homeostasis, which is ensured by the coordinated participation of many organs, hormonal systems, and specific hormones (cerebral cortex, hypothalamus, pituitary gland, adrenal cortex, kidneys, lungs, stomach, intestines, ACTH, ADH, aldosterone, renin, etc.). All this makes it untenable to assess the total supply of the body with one or another metal by its content in the hair. At the same time, the very fact of a power-law connection between the content of metals in hair and the number of individuals (a sign of criticality) deserves attention, since the fractality inherent in this relationship (independence of the scale of the system) makes it possible to identify criticality at any level suitable for such an analysis. In other words, if there really is a power-law connection between the number of cells and the content of metals in them, then it should obviously manifest itself not only at the level of cells, but also at the level of individuals. This greatly simplifies the research task and, as our experience shows, makes it quite solvable with the help of hair spectrometry.

The authors are grateful to the staff of the Center for Biotic Medicine (Moscow), first, A.R. Gabelli’s, for kindly providing a database of Na, K, and Ca hair mass spectrometry using a NexION 300D instrument (Perkin Elmer Inc., Shelton, CT, USA).

1. Marshall WJ (2000) Clinical biochemistry. BINOM-Nevsky dialect, Russia, pp. 448.
2. Petukhov VI, Dmitriev EV, Baumane L (2019) Homeostasis of sodium (Na) and potassium (K) in epidermis as a self-organized criticality phenomenon. Proteomics & Bioinformatics 2(1): 62-67.
3. Petukhov VI, Dmitriev EV, Baumane L, Skalny AV, Lobanova YN (2016) Electrogenic metals in epidermis: relationship with cell bioenergetics. Insights in Biomed 1(2): 9-14.
4. Petukhov VI (2017) What are the limits, if any of normal content of electrogenic metals (K, Na, Ca) in epidermis? Insights in Biomed 2(2): 13-7.
5. Petukhov VI, Baumane L, Dmitriev ЕV, Vanin AF (2014) Nitric oxide and electrogenic metals (Ca, Na, K) in epidermic cells. Biochemistry (Moscow) Supplement Series B Biomedical Chemistry 8(4): 343-348.