CONTENT OF TRACE ELEMENTS, TOXIC METALS, VITAMINS,

AMINO ACIDS AND/OR HORMONES

DESCRIPTION

Summary of the Invention

The present invention relates to a method for assessing the metabolic functions of an individual through the analysis of the hair bulb and more specifically an analysis indicative of the intracellular conditions, i.e., of the various metabolisms and activities occurring inside the cell (for example at the mitochondrial level), through the determination of the content of trace elements, toxic metals, vitamins, amino acids and/or hormones, capable of providing better functional diagnosis and biological therapy possibilities.

According to the invention, this method exploits the combination of remote sensing and deconvolution in a diagnostic key, using: - fluorescence microscopy with darkfield polarized light refraction with a high- resolution digital camera as detector, which allows the acquisition of Full HD,

3D images of the hair bulb, and

- the software already disclosed and for which a registration request has been made in Italy with the Special Public Register for Computer Programs, referred to as Ale. 19 by the same Applicant, implemented on a PC, which allowed to read and transform a broad spectrum of color frequencies into objective data.

In particular, a state-of-the-art vertical microscope was used such as the Leica DM6 B, which is provided with a light source for fluorescence excitation with polarization contrast and a color high-resolution digital camera such as the DFC 495 as detector.

The integrative comparison of the various tests carried out with this method thus becomes a procedural method capable of effectively and sensitively detecting and quantifying a large number of trace elements, vitamins, amino acids, hormones and so on, for the definition of functional metabolic profiles, through the simple analysis of the hair bulb, impossible until today.

General concepts and background art - Definitions

For the sake of clarity, the meaning of a series of terms used in the present description and which refer to the field of spectral analysis and digital image processing, according to the current state of the art, are provided below.

A) Remote sensing means the technique which allows to obtain qualitative and quantitative information on objects (samples) placed at a distance from a sensor (camera), by means of measurements of electromagnetic radiation emitted, reflected or transmitted by the surfaces under examination, without coming into direct contact. The measurements of the electromagnetic radiation, which interacts with the physical surfaces of interest, are provided in the form of high-resolution images, and detected by special remote sensors. Each element has a well-coded and globally-known spectrum (solid and liquid refractive index scale).

In this specific case, all the elements present in the hair bulb and follicular stem are optically active and birefringent substances.

In order to best exploit the richness of the information contained therein, the images are not shown in levels of gray, but by means of the use of color with polarized light, through the principle of the additive synthesis of the three basic colors RGB, red, green and blue. The maximum intensity value of the three primary colors corresponds to white and the minimum to black.

The polarized light passes through the bulb for the entire thickness thereof, emitting and displaying a colored map containing the various wavelengths. The color which characterizes an element, whether vitamin, mineral, amino acid or hormone, is produced by the selective absorption of certain wavelengths by atoms belonging to the structure of the element itself. It is therefore possible to obtain biochemical and morphological information from these multispectral images acquired in different bands of the electromagnetic spectrum. Remote sensing is therefore an applied science with diagnostic- investigative purposes which allows to identify, measure and analyze the qualitative and quantitative features of a given object, without coming into direct contact, offering complete biochemical and morphological information. B) In digital image processing, the term "convolution" indicates a mathematical correction technique to define the value, weight, membership of a sample based on the application, on the same image, of a specific algorithm, such as PCA (Principal Component Analysis), ICA (algorithm for multi-variant signal separation), SCM (algorithm for spectroscopy analysis for data definition), IDL (scientific programming software to analyze and process multi-spectral images).

In the present application, spectral unmixing is used to break down the reflectance of the spectrum and the measurements relating to the same area acquired simultaneously.

A multispectral image is the composition of a high number of pixels and spectral unmixing is an algorithm applied to fluorescence images to correct the overlapping between the emission spectra of the fluorochromes (spectral bleed-through) between the different color channels. For example, the emission spectra of CFP and YFP fluorochromes partially overlap and CFP signals even appear in the YFP fluorescence channel. This overlapping can be corrected by spectral unmixing.

Furthermore, the use of spectral unmixing allows to determine the biological composition of a sample depicted in a spectral image with different wavelengths present. Spectral indices are the most effective means for monitoring the various samples and are the result of mathematical operations between the reflectances of the surfaces investigated.

Unmixing is even used to break down a reflectance of the source spectrum into a series of data endmember spectra and therefore serves to support all the various processes to define the various values.

Endmembers are defined as materials which are spectrally unique in the wavelength bands used to collect the image, i.e., the spectra of the final endmembers cannot be reconstructed as a linear combination of other image spectra.

The result of spectral unmixing is a measurement of the composition of the individual endmembers at the source spectrum. This measurement is referred to as endmembers. The selection and reading of the electromagnetic radiation and of the endmembers present in the various pixels occurs by means of some algorithms listed above. The spectral action of the unmixing allows to precisely calculate, through quantum algorithms, the diffraction spectrum on the single element, membership and various measurements.

This method is part of complementary analytical techniques such as polarized light microscopy.

The procedure we have adopted always allows us to precisely find the wavelength (on birefringent elements), which cannot occur with destructive diffractometric analyses, because the presence of an element is determined only in particular concentration and temperature conditions. Furthermore, this method allows the reproduction of the test on the same sample with always identical results.

Therefore, we can additionally exploit this method by analyzing quantities of spectra in physically connected regions within the image, allowing the reconstruction of a sub-pixel-graphs by combining and associating the weight of both sub-graphs and therefore without breaking down, separating or altering the same sample.

C) A Leica DM6 B microscope was used as the optical media, configured to operate in a darkfield with polarized light, fully motorized, which allows to automate the scanning operation of the slide on all three axes, x, y and z, as well as the complete management of optics and lighting by means of PC. Sharper and more precise digital images are obtained on the same PC connected to the optical microscope.

D) The darkfield microscope is obtained by placing a darkfield diaphragm under the condenser lens system of an optical microscope. A hollow cone of light is thereby created which hits the object and only the reflected and refracted light from the sample will form the image.

Thus the sample cells will appear multi-colored, depending on the birefringence thereof, on a dark background.

This technique allows to improve the contrast differences between the cells and the surrounding medium, avoiding the use of stains.

Developed by Enderlein and widely used in hematology, darkfield microscopy allows a very significant morphological view: for example, the shape, size and behavior of blood cells can be indicative for the dynamic study of an organism's biology.

When natural light is passed through a polarizing filter, it selects the waves based on the plane of vibration thereof. The resulting light, while remaining composed of waves which vibrate on various frequencies, vibrates only on one of the infinite planes on which the natural light vibrates. In fact, it should be noted that natural light not only oscillates on the different frequencies corresponding to the light spectrum colors from ultraviolet to infrared, but these oscillations occur on all planes perpendicular to the direction of the light propagation. The first of the two polarizing filters is placed between the light source and the sample containing the crystals. The second, called analyzer, is instead placed between the sample and the observer.

The two polarizers are oriented with respect to one another, so that if the first allows only the vertical oscillations to pass, the second, rotated 90° with respect to the first, allows only the horizontal oscillations to pass. The light would consequently be entirely obscured, if the crystalline lattices, which are the typical object of observation with this microscope, were not optically active and did not rotate the light polarization plane by a certain angle. The light is thereby not entirely blocked by the second filter, which rather allows to analyze the particular modification to which the light has been subjected by the crystal.

In fact, most crystals present the birefringence phenomenon, i.e., they are able to split polarized light into two radiations which oscillate at different frequencies, one on an orthogonal plane with respect to the plane on which the other oscillates.

The analyzer filter is thus reached by the light which has passed through the crystal and is not completely lacking the horizontal component, because it has undergone a further polarization. This light is colored as well, because one ray has a different frequency than the other and the frequencies of one interfere with those of the other. Polarized light microscopy has been the traditional domain of mineralogy from the very beginning, but the usefulness thereof in the field of trichological and trichychiatric science is now beyond question. In fact, tissues such as keratin, whether in alpha or beta, offer a field of investigation in polarized light.

Context of the Invention

From the early studies of Dr. Clivet, to date, various scholars and research groups have allowed to correlate the presence of trace elements in hair with the nutritional conditions of the organism. In particular, it was possible to determine the metabolic balances or imbalances, i.e., the intra-cellular enzymatic reactions, and various physiological states of malaise including metabolic disorders, physical fatigue, mental and emotional conditions, glandular disorders.

Considered as a biological sample for use in diagnostics, the hair bulb shows in the structure thereof the history of human minerals, nutrition and lifestyle of the subject.

The hair bulb is formed in the dermis by various cellular chains referred to as "matrix" which is at the base of the follicle. The anagen-catagen-telogen cycle and terminal hair differentiation is controlled by sex steroids and glucose metabolism, while the multiplication of hair matrix cells and keratin synthesis are likely under the control and interaction of two growth factors, an HRGF2 stimulant and an EGF and/orTGF inhibitor.

The stacking and cellular stratification of hair which occurs in the active parts allows us to know and monitor the body's adaptation to the harmful events to which it is subjected daily.

During the growth phase the metabolic activity of the matrix is very complex and high, thus the hair can be affected by contingent physiological and pathological variations. When the hair reaches the skin surface, the external part thereof hardens, thus "crystallizing" the metabolic products accumulated during the formation of the hair itself. Growing about a centimeter every month, hair even concentrates more than a hundred times the trace minerals, organic compounds and metabolic products present in the circulating liquids and retains them.

The possibility of determining a mineralogram is mostly based on the Hair Tissue Mineral Analysis - HTMA assay, where the hair is chemically treated under drastic conditions, incinerated and analyzed in plasma spectrometry. A work published in the Journal of the American Medical Association and highlighted by the American body FDA (Food and Drug Administration) shows that these procedures can alter the content and certainly demonstrate that the HTMA test is a non-repeatable test, as the hair is exposed to very high temperatures (up to 10,000 °C) and then destroyed. The analysis is provided only on the stem and not on the bulb, providing altered results and only exogenous results, therefore the test is only capable of determining the presence of minerals and not of heat-labile organic substances, such as for example vitamins, amino acids and hormones, which are destroyed in the heat treatment of the analysis.

In the European patent application EP 2 518 474 describes a method for the determination of hair analysis, which method involves using polarized light not only to provide information on the mechanic anatomical and intracellular structure of the hair, but also to obtain qualitative and quantitative information on the chemical components of the hair, with special reference to the analytes of interest, such as metals, amino acids, drugs, hormones, vitamins, pollutants, poisons, etc.

In conclusion, the currently known method comprises, in successive steps: a) (calibration step) constructing a light pattern for each pure analyte (final elements); b) (measurement step) constructing a light pattern of the hair sample under analysis; c) (determination step) comparing the models obtained in a) with the models obtained in b) and determining the relative contributions of each analyte in the hair sample, by means of a software - assisted by unmixing processing.

However, this method is not totally reliable since it is essentially based on digital microscope images which are not always of the best quality and where disturbing elements may be present. Furthermore, the resolution of the 2D images does not allow to correctly assess the real quantities of the individual analytes in all the hair portions.

The method mentioned above is based on software (IDL), the observed spectra processing power of which was however limited and based on the transformation of spectra based on polarized light.

There is again a need for an analysis method of the hair and the bulb thereof capable of determining the analytes of interest qualitatively- quantitatively and with the greatest possible sensitivity and reproducibility. Detailed description of the invention

The advent of new instruments, with greater resolution power, and the introduction of fluorescence microscopy, has increased the potential of hair analysis for the determination of the analytes present.

However, this involves the re-determination of all required standard references and the spectra of the reference analytes. Furthermore, the amount of data and potentially obtainable spectra require a software with greater processing and integration power. The aim of the invention is to overcome these limits and drawbacks, again resorting to the combination of remote sensing and deconvolution but using on the one hand a state-of-the-art digital microscope such as the Leica DM6 B with polarized light, in fluorescence, which allows us to have Full HD, 3D images of the hair bulb, and on the other hand, a software available to the same Applicant, already disclosed with the name ALE.19, which allowed to read and transform a broad spectrum of color frequencies into objective data.

The use of fluorescence spectroscopy arises from the following observation: all spectroscopies consist of measuring the interaction between energy and matter. The absorption of energy by a molecule can cause chemical and/or physical variations (properties of the electrodes or of the nucleus) of the chemical type. The absorption or emission can provide information on the structure of the molecules and/or the variations it undergoes.

Fluorescence spectroscopy has advantages and disadvantages, but combined with polarization it ensures the possibility that all molecules emit significant signals even at very low concentrations. Thereby, the test object of the present invention referred to as "Biological Hair Bulb Research" or "BHBR" was perfected, which reads in mg% corresponding to mg. 0.000001. The absorption of light by a molecule corresponds to a new energy which promotes an excited electron (Resonance Energy Transfer), while the horizontal polarization allows to visualize the membership bands with the spectral colors.

Advantageously, according to the invention, the images are again read by leaving the polarizer in darkfield; this very important datum allows to have a fixed starting parameter and to always have comparable parameters. The samples used for the test do not undergo any physical or chemical manipulation, thus differentiating themselves from any other similar examination.

Each element has a well-coded and globally-known spectrum (solid and liquid refractive index scale) and all the elements present in the hair bulb and follicular stem (vitamins - nutritional and toxic minerals - amino acids - neurotransmitters - hormones) are optically active and birefringent substances. Crystals are particles characterized by a well-organized molecular arrangement. They generally have three optical axes which correspond to the particle length, width and depth. These organic substances have the prerogative of rotating the plane of polarized light. The optical rotation measurement can be used both for recognizing a certain substance and for quantitative determination. An optically active substance can cause the plane of polarized light to rotate right or left. In the first case it is called dextrorotatory, in the second case levorotatory. Normally a chinal substance (elliptical shape) originates two optically active isomers, called essentiomers or optical antipodes, which are the mirror image of each other. Therefore, in order not to alter or modify the birefringence, refraction and wavelength, the polarizer always remains fixed in darkfield.

The potential and results offered by this method are remarkable if the relative ease of obtaining information of any kind in a short time and at a distance (without touching the sample) is considered. This therefore represents a real revolution in the analytic field.

Advantageously, the use of fluorescence-refracted polarized light microscopy allows to simultaneously capture all the frequencies of the various elements of the analyzed sample.

The integration of the use of fluorescence-refracted polarized light microscopy, with the possibility of obtaining very high resolution and 3D images, and with the greater processing power/resolution of the software ALE.19, has allowed to determine the new values and spectra for the reference analytes, which form the basis of the new test object of the present invention, BHBR.

In particular, this BHBR test (Biological Hair Bulb Research) is a polymetabolic test which differs from the usual blood tests in that it reveals the real intra-cellular situation of intracellular metabolic processes, while blood tests are mainly in relation to the transport of minerals to and from the intracellular areas. The BHBR should be considered as a screening test. The examination of hair, due to the biological features thereof, offers a complementary tool to routine analyses, such as blood and urine analyses. If the blood and urine analyses offer a current picture of the state of the organism and therefore provide information on the existing pathological situation, bulb analysis is instead able to tell the metabolic "history" of the presence of minerals, therefore allowing to cast a broader and more predictive view of the previous conditions which generated the present situation and which could generate metabolic or pathological disorders.

The BHBR test which is described is therefore indicative of the intracellular conditions, i.e., the various metabolisms and activities occurring inside the cell (for example at the mitochondrial level). In blood analyses, the levels of minerals and other active ingredients are generally maintained by taking them directly from the tissues, are greatly influenced by changes in the various metabolisms and emunctorial functions and are subject to changes and fluctuations in the same day or over short periods of time. In the anatomical structures of hair, the intracellular content is instead a stable value which allows to investigate and detect a considerable amount of information which has direct relevance to the health and efficiency conditions of organs, systems and body systems.

Thus the integrative comparison of the various tests with BHBR becomes a procedural method capable of providing further and better possibilities for functional diagnosis and biological therapy. Values, data and ranges were obtained by investigating over 13,850 subjects for research purposes.

The method followed is the protocol disclosed by the Clinical and Laboratory Standards Institute (CLSI) to determine the frequency intervals on a healthy population. The data were validated by comparison through the golden standard with ICP-MS (Inductively Coupled Plasma Mass Spectrometry) carried out at the Department of Medicine of the University of Udine, Prof. Curcio.

Unlike the tests used so far, the greater resolution power at the base of BHBR is able to detect intracellular activity. The inside of the cell is in fact the place where the most important metabolic phenomena occur. The examination is based on the interpretation of the intracellular content of the bulb and exploits the role thereof as a "recorder" over time. Due to the greater resolution power thereof and greater sensitivity in determining the individual analytes, the BHBR test is able to simultaneously capture all the frequencies of the various elements of the analyzed sample and an individual biochemical identikit on the function of the different metabolisms, highlighting which balances are in altered conditions. The active parts of the bulb and stem are analyzed with the assessment of the biochemical and metabolic components of the hair. The test investigates the intracellular elements both in terms of nutritive and toxic minerals, as well as vitamins, hormones and amino acids. The examination, intended as "screening", can therefore be extremely useful if combined with normal clinical examinations, with diagnostic assessment judged by the treating physician. In conclusion, this test provides four important pieces of information:

Assessment of the overall mineral balance and identification of the endocrine and autonomic state of the organism

Detection of any accumulation of toxic minerals

Assessment of the action of vitamins, amino acids and organic defenses

Real-time assessment of the metabolic state in progress with the possibility of subsequent examinations which show the adaptations and compensations that have occurred.

As reported here, the term "ideal value" is the ratio between two minerals which, according to Watson and Eck, represents the best metabolic balance between the two elements: this value must be the therapeutic indication for the doctor. If the value found is low or high compared to the ideal value, it can be considered an "unbalanced" ratio and defined as "mild, medium or severe" and considered a predictive index of various pathological trends.

As reported here with the term "ratio" between two minerals, synergy is defined as the homeostasis reaction between two minerals which must complete each other in the functions thereof as well. The ratio between two elements has the same or even greater importance than the level of the single element. The ratio between two minerals is calculated taking into account the values of each single element, the extension of the single interval and the ideal value of the ratio itself. Therefore, it is possible to have two minerals comprising the ratio with normal values, while the ratio found can be high or low compared to the normal value.

As reported here with the term "predominance", in the ratio between two elements, it is precisely indicated which dominates the same ratio, taking into account the range value of the single mineral. A predominance between two minerals (i.e., excess) of one mineral in ratio to another may sometimes occur even when the two individual minerals are below the respective normal values thereof.

Unlike what is reported in the state of the art, and unlike patent document EP 2 518 474, the BHBR test with the values and graphs thereof of minerals (toxic and nutritional), vitamins, amino acids and hormones, reflects all the levels of a person's various functions. The intracellular biochemical components of the hair's metabolism indicate physical state, body biochemistry, diet, lifestyle. The BHBR analysis carried out on the hair structures, in the active parts of the bulb and the stem, is capable of showing the adaptations and compensations occurring in the body, particularly at the cellular level.

As a general diagram, the method according to the present invention includes the following steps:

I) fixing the sample, i.e., the hair consisting of bulb and stem, arranged in an aligned manner, on a microscopy slide using a colorless fixative (natural Canada balm);

II) irradiating with polarized light in fluorescence using a microscope such as the Leica DM6 B model;

III) obtaining a digital image of the hair bulb in full HD and three- dimensional 3D using a high-resolution camera as detector;

IV) constructing a light pattern according to the modalities illustrated below which form a peculiar feature of the present invention, and comparing by means of ALE.19 software the light model thus constructed with those obtained for the reference analytes by means of an assisted mixing processing which allows the related contribution of each analyte present in the bulb sample to be determined;

V) determining the absolute quantity and/or concentration of the identified analytes in the hair sample (ppm, pg/mg, etc.).

According to the invention, the light patterns referred to in step IV are constructed starting from the Full HD 3D digital image obtained by the high- resolution camera connected to the DM6 B microscope analyzing the sample (pure analyte or hair bulb) in darkfield-fluorescence refracted polarized light in the following ways: a matrix of points is constructed from this image, where each point corresponds to a single pixel of the image, and is defined by the wavelength and absorbance value of said pixel; where all the pixels of the image are included in a matrix; and where the matrix comprises all possible pixel wavelengths, i.e., the sampling is not limited to the discrete wavelengths selected.

As a result, very precise light patterns are obtained from standard analytes and hair samples.

Through the software ALE.19, the matrix data are then processed via assisted mixing software: the use of an unmixing algorithm results in the related contribution of each individual analyte to the overall digital image of the hair sample, providing a very precise and reproducible assessment of the analytes in the bulb, comprising those present in trace amounts.

According to a peculiar feature of the invention, the analysis of the hair bulb thus conducted shows the adaptations and compensations occurring in the body, allowing to provide an assessment index of the oxidation rate of a person who can be a hyper-oxidizer or hypo-oxidizer, which is an index of stress.

For example, the Sodium-Potassium ratio (with a range from 2.0 to 9.0 and an ideal value of 2.5:1) and the Calcium-Potassium ratio (with a range from 2.0 to 9.0 and an ideal value of 4.0) can allow to assess the oxidation rate and the stress phase. The oxidative rate measured in the hair analysis is the sum of the thyroid and adrenal ratio according to the oxidative typologies acclaimed by Prof. George Watson, Dr. Hans Seyle, Wilson and Eck (slow oxidizers and fast oxidizers). Activated sympathotonic stimulation is accompanied by an increase in the speed of cellular oxidation with an increase in thyroid and adrenal reactivity.

In essence, with acute stress we will have high Calcium-Potassium and Sodium-Potassium values, while with chronic stress we will have very low Calcium-Potassium and Sodium-Potassium values.

Experimental Part

Applying the above method, the reference values were determined for the analytes of interest to use as reference. The data, values and intervals were processed with the CLSI (Clinical and Laboratory Standards Institute) protocol and validated by the Golden Standard ICP-MS (Inductively Coupled Plasma Mass Spectrometry) at the Department of Medicine of the University of Udine.

Below are the reference values for trace elements, toxic minerals, vitamins, amino acids and hormones. The values are expressed in mg%, which correspond to mg 0.000001.

Trace elements The reference values for trace elements are shown in Table 1

TABLE 1

Toxic Minerals:

The reference values for toxic metals are shown in Table 2.

TABLE 2

Vitamins:

The reference values for toxic vitamins are shown in Table 3

TABLE 3

Amino acids:

The reference values for amino acids are shown in Table 4 TABLE 4

Hormones:

The reference values for hormones are shown in Table 5 TABLE 5

Relations/Ratios

With the term ratio between two minerals, synergy, or combination, is defined as the homeostasis reaction between two minerals which must complete each other in the functions thereof as well. The ratio between two elements has the same or even greater importance than the level of the single mineral. The ratio between two minerals is assessed and interpreted through the range thereof and the ideal value thereof and is predictive or/and indicative for important parameters. For a global interpretation of the test, it is useful to use normal clinical examinations as well. In this case, we can have information regarding:

Protein and "Vitality" metabolism in the Calcium-Potassium, Sodium- Magnesium, Sodium-Potassium ratios; for a possible assessment of the energy activity, of the oxidation rate, as indicators of tolerance to carbohydrates and for an assessment of stress. Thyroid Function in the Calcium-Potassium and Zinc-Copper ratios for an indication of the thyroid cell activity, for an assessment of the stress phase and energy efficiency according to Dr. Hans Seyle and Dr. L.D. Wilson.

Glycidic Metabolism in the Calcium-Magnesium and Sodium-Potassium ratios can be indicative of the function of autonomic balance, correct neuromuscular function and a possible sensitivity to glucose, according to L.D. Wilson.

Neurovegetative Balance in the Calcium-Phosphorus ratio to assess the subject as a hypo-oxidator with a predominance of Calcium, or a hyper- oxidator with a prevalence of Phosphorus according to Dr. P.D. Wilson and Dr. Paul Eck.

Neuropsychic Stability in the Calcium-Sodium ratio for an assessment of normal adrenal activity according to Dr. Hans Seyle. Immune system balance in the Sodium-Potassium, Zinc-Iron ratios may indicate a weakening of the immune defenses according to Dr. Paul Eck. Female hormonal balance in the Zinc-Copper ratio may be indicative of correct functions in the female hormonal system. In fact, copper is connected to estrogen secretion, while zinc is involved in the synthesis of progesterone. Hormonal Profile in the Zinc-Iron ratio can be indicative of hormonal balance, for the immune system and to assess normal protein synthesis.

Insulin balance in the Chromium-Vanadium ratio is an assessment from which glucose intolerance may derive.

Balance and Energy Function in the Calcium-Phosphorus, Calcium-Potassium, Sodium-Magnesium, Zinc-Copper ratios can be indicative for an assessment of the thyroid and adrenal glands function, responsible for energy production.

The presence of toxic metals such as Cadmium, Mercury and Lead could interfere with energy production, as indicated by Dr. L.D. Wilson, Dr. Paul Eck. Intolerance and sensitivity in the ratios: Sodium-Magnesium, Zinc-Copper, Iron-Copper, the presence of toxic metals, the state of normality of Histidine, Noradrenaline, Tryptophan and B vitamins, are indicative for a possible assessment of the individual in question, who may have food sensitivities.

Below are the ratios of minerals and the physiological-biochemical correlations thereof. The reference values refer to different types of people: Child from 3 to 12 years (Child); Man/Woman (M/W); Man over 60/Woman after menopause (M+60/Wm).

Ratio: Sodium/Magnesium - Adrenal Profile

The Na/Mg ratio is used to investigate the cortical function of the adrenal glands, as these glands control Sodium levels. Indicates balance between mineralocorticoids and corticoids, provides information on the oxidation rate and energy efficiency.

Ideal Value 4.16 - 4.17:1 Normal range 2.25 - 9.79:1 M+60 Wm Normal range 2.00 - 9.70:1 MW Normal range 1.76 - 7.06:1 Child

Zinc/Iron Ratio - Hormonal Profile

Allows to assess normal protein synthesis and the immune system. Zinc is essential both for the functioning of over one hundred enzymes as well as for the persistence thereof. It plays an important role in cellular reproduction, it is the true reflection of the metal's organic availability. An iron deficiency can be highlighted in hair before significant changes in hemoglobin occur.

Ideal Value: 5.7:1

Normal range 0.73 - 8.50:1 M+60 Wm Normal range 1.40 - 9.00:1 MW Normal range 0.59 - 7.90:1 Child

Zinc/Copper - Immune Profile

Indicative for the functional state of hormones, particularly female, for the function of the immune system, for the metabolism of cholesterol and lipids. The balance between Copper and Zinc is very delicate and important; in fact, when Zinc is deficient, Copper tends to accumulate in some organs.

Ideal Value: 8.0:1

Normal range 1.10 - 11.00:1 M+60 Wm Normal range 1.39 - 12.00:1 MW Normal range 0.53 - 9.50:1 Child

Iron/Copper - Infection receptivity profile

Allows to assess the predisposition to viral and bacterial infections as well as the inflammatory state with anemia. Ideal Value: 1.40:1

Normal range 0.90 - 4.05:1 M+60 Wm Normal range 0.90 - 4.10:1 MW Normal range 0.70 - 4.00:1 Child Calcium/Magnesium - Carbohydrate profile

Carbohydrate and sugar and carbohydrate tolerance profile. Provides information on the function of glucose metabolism, neurovegetative balance as well as neuromuscular activity and glucose sensitivity.

Ideal Value: 6.67:1

Normal range 5.00 - 13.50:1 M+60 Wm Normal range 4.60 - 12.70:1 MW Normal range 4.01 - 10.50:1 Child

Calcium/Phosphorus - Neurovegetative and oxidative metabolisms profile.

This ratio allows to assess the neurovegetative balance and oxidative metabolisms; if Calcium prevails in this ratio, the subject is parasympathetic dominant, therefore a hypo-oxidator. If Phosphorus prevails, the subject is experiencing sympathetic neuroendocrine dominance, therefore a hyper- oxidator.)

Ideal Value: 2.50 - 2.62:1

Normal range 2.20 - 27.00:1 M+60 Wm Normal range 1.30 - 23.00:1 MW Normal range 1.80 - 19.50:1 Child

Calcium/Potassium - Thyroid profile

It is an assessment index of the oxidation rate, an index of stress, energy efficiency and thyroid activity at the cellular level. The Ca/K ratio is a good indicator of the peripheral activity of the thyroid hormone.

Ideal Value: 4.0 - 4.2:1 Normal range 3.20 - 21.50:1 M+60 Wm Normal range 2.00 - 19.00:1 MW Normal range 3.00 - 15.50:1 Child

Calcium/Sodium - Neuropsvchic profile

This ratio allows to assess hypo and/or hypertensive states

Ideal Value: 1.60 - 1.75:1 Normal range 1.00 - 43.30:1 M+60 Wm Normal range 0.80 - 36.30:1 MW Normal range 0.90 - 32.00:1 Child

Calcium/Zinc - Lipid profile

This ratio allows to assess the cortico-hypothalamic axis, lipid metabolism, glucose metabolism. When the ratio is high, it indicates clear dysfunction.

Ideal Value: 2.0:1

Normal range 1.80 - 36.50:1 M+60 Wm Normal range 1.20 - 31.50:1 MW Normal range 1.10 - 26.50:1 Child

Sodium/Potassium - Protein profile

The ratio expresses an assessment of adrenal activity, like the Na/Mg ratio. It provides information on protein metabolism and stress conditions as well. Ideal Value: 2.5-1

Normal range 1.30 - 12.50:1 M+60 Wm Normal range 1.25 - 11.50:1 MW Normal range 1.30 - 9.90:1 Child

Chromium/Vanadium - Insulin profile

The ratio allows to assess insulin balance. An imbalance in the insulin ratio may indicate the presence of an insulin resistance pattern from which glucose intolerance may arise. In the Chromium/Vanadium ratio, Chromium forms the extracellular insulin receptor, while Vanadium forms the intracellular receptor.

Ideal Value: 0.5 - 1

Normal range 0.10 - 1.00:1 M+60 Wm Normal range 0.10 - 1.10:1 MW Normal range 0.10 - 1.18:1 Child

Calcium/Vit.D - Parathyroid profile

Function of the parathyroid glands, balance of calcium, magnesium and bone metabolism. Function of the nervous and muscular systems and energy metabolism.

The ratio indicates the functional ability of the parathyroid glands and clarifies the efficiency thereof in managing the parathyroid hormone, which regulates the metabolism of calcium and phosphorus and of the bones.

Ideal Value: 21.33

Normal range 25.35 - 76.05:1 M+60 Wm Normal range 21.26 - 63.79:1 MW Normal range 18.26 - 54.79:1 Child The new Framework consists of a series of new ratios which have been created after in-depth research, with the aim of describing the various gradualities and differentiations in the completion phases of the various metabolic processes. These ratios have been identified for the role and functions which the comprised elements perform within the organism. It is important to note that the ratios indicated below comprise groups and series of substances which, within an organ or apparatus structure, and the whole organism as an overall biological entity, will carry out activities, which may vary from synergy to antagonism in reference to the different phases and to the biochemical and intracellular processes in which they are involved during the different biological and physiological cycles. It is therefore important to keep in mind that the predominance of one or the other element or group will not necessarily have different clinical manifestations but may, for these reasons, result in the same conditions, the same disorders and dysfunctions.

1 Mineral/Mineral Unit Ratios

Sodium/Potassium-Calcium/Magnesium - Lymphatic profile

An altered ratio, predominantly of one or the other comparison group, will reveal a poor efficiency of the lymphatic system which may be the cause of disturbances such as allergies, sinusitis, eczema and skin problems in general, excessive sweating, cellulite, being overweight and obesity, ear problems, neck pain, tissue edema, chronic fatigue, repeated viral, bacterial and parasitic infections, migraines and headaches, even severe changes in the immune system such as some forms of arthritis and fibromyalgia.

Ideal value: 23.93

Normal range 18.98 - 56.94:1 M+60 Wm Normal range 15.39 - 46.18:1 MW Normal range 13.50 - 40.51:1 Child Calcium/Zinc-Chromium/Vanadium - Leptin index

An altered ratio, predominantly of one or the other comparison group, may indicate leptin deficiency and/or leptin resistance, which may lead to conditions of amenorrhea, infertility and osteopenia and osteoporosis.

Ideal Value: 12.90

Normal range 13.34 - 40.02:1 M+60 Wm Normal range 11.41 - 34.25:1 MW Normal range 9.64 - 28.94:1 Child

Calcium/Potassium-Sodium/Magnesium-Membrane Index

Important ratio for assessing cell membrane permeability and the efficiency of the active - primary and secondary - and passive transport of molecules. An altered ratio indicates an altered cell membrane physiology in the lipid and protein components thereof, resulting in an imbalance in passive diffusion and active transport.

Ideal Value: 24.68

Normal range 18.98 - 56.94:1 M+60 Wm Normal range 15.39 - 46.18:1 MW Normal range 15.00 - 45.01:1 Child

2 Mineral/Mineral Unit Ratios Zinc/Iron-Sodium/Potassium - Resilience index

The ratio is interpreted in the light of the knowledge of psychology, which with the term "resilience" defines the ability to face traumatic events in a positive manner, to positively reorganize one's life in the face of difficulties, and therefore know how to start over and rebuild with an open, well-disposed attitude, being able to accept and seize new positive opportunities which life can offer while keeping one's own humanity. Ideal Value: 8.95

Normal range 6.62 - 19.86:1 M+60 Wm Normal range 5.81 - 17.44:1 MW Normal range 5.48 - 16.44:1 Child

Calcium/Magnesium-/-Calcium/Phosphorus - Buffer systems profile

Through the synergy and antagonism relationships of the elements comprised, this profile allows an assessment of the efficiency of the buffer systems (intracellular, extracellular and bone) and allows to deepen aspects related to bone/joint structure health.

Ideal Value: 25.75

Normal range 26.98 - 80.95:1 M+60 Wm Normal range 22.39 - 67.18:1 MW Normal range 19.29 - 57.89:1 Child

Sodium/Magnesium-Chromium/Vanadium - Persistence profile

The term "persistence" means a condition which lasts over time, is continuous and constant. The ratio explores the aspects relating to the energy metabolic framework and highlights particular conditions of the functional aspects in the relations of synergy and immune neuroendocrine antagonism. By intervening in different phases of the different metabolisms, not least that of carbohydrates and lipids, the ratio defines the functionality of the set of mechanisms in which adrenaline, glucagon and insulin and enzymatic complexes such as trypsinogen, chymotrypsinogen, lipase, amylase are primarily involved.

Ideal Value: 5.10

Normal range 8.01 - 14.12:1 M+60 Wm Normal range 12.22 - 18.33:1 MW Normal range 2.78 - 8.36:1 Child

Function group sub-unit 1 Minerals/Vitamins Phosphorus/Vitamin B12 - Psycho-emotional index

The emotional index comprises the management of emotions, meaning the mental and physiological states associated with certain psychophysiological changes due to internal or external, innate or learned stimuli.

Ideal Value: 2.82

Normal range 2.05 - 6.15:1 M+60 Wm Normal range 2.07 - 6.21:1 MW Normal range 1.52 - 4.58:1 Child

Iron/Vitamin C - Basal index

The basal index should not be confused with basal metabolic rate, which is only one aspect. Useful in order to investigate problems of hemoglobin formation, iron metabolism, thyroid function stimulation, free radicals and reactive types and antioxidant systems, immune capacities against viruses and bacteria.

Ideal Value: 1.83

Normal range 1.34 - 4.04:1 M+60 Wm Normal range 1.32 - 3.96:1 MW Normal range 1.00 - 3.01:1 Child

Copper/Vitamin B3 - Coenzyme coefficient

The term enzymatic cofactor (or coenzyme) refers to the substance which consists of the non-protein portion of the enzyme, defined as the prosthetic group. The correct functioning of the chemical reactions catalyzed by enzymes requires that the protein and non-protein portion join together to form a complex. Although many enzymes can consist of a simple protein (these are defined as unitary constitution enzymes), many others require the addition of the aforementioned prosthetic group to the protein, often consisting, for example, of vitamins (such as those of the B group) but even of metal ions. In these enzymes, which are said to have a dualistic constitution, the complex takes the name of holoenzyme, of which the protein consists of the apoenzyme and the prosthetic group consists of the coenzyme.

Ideal Value: 2.76

Normal range 2.02 - 6.06:1 M+60 Wm Normal range 2.03 - 6.09:1 MW Normal range 1.47 - 4.42:1 Child

Selenium/Vitamin A - Antioxidant profile

Antioxidants are molecules which slow down or prevent the oxidation of other molecules, thereby inhibiting the production of free radicals. Selenium is defined as an "antioxidant" for the powerful ability thereof to counteract the formation of free radicals in the body. Selenium is important in order to assess the efficiency of the immune system, of the antioxidant systems and capacities, it is linked to the peripheral enzyme deiodinase which is synthesized in the liver and kidney and is essential for the proper function of the thyroid gland, required for the conversion ofT4 into T3.

Ideal Value: 5.26

Normal range 3.80 - 11.40:1 M+60 Wm Normal range 3.85 - 11.57:1 MW Normal range 2.86 - 8.58:1 Child Function group sub-unit 2 Vitamins/Amino acids Vitamin C/Lysine - Tissue profile

Provides general information on connective tissue and in particular on collagen.

The human body is composed of four basic types of tissues: epithelial, connective, muscular, nervous.

Ideal Value: 1.08

Normal range 0.79 - 2.38:1 M+60 Wm Normal range 0.75 - 2.25:1 MW Normal range 0.62 - 1.86:1 Child

Vitamin D/Histidine - Inflammatory profile

The term inflammation, or phlogosis, defines an innate and non-specific defense mechanism, which consists of a protective response following the harmful action of physical, chemical and biological agents and the final goal of which is the elimination of the initial cause of cell or tissue damage, as well as the activation of the reparative process. The ratio provides information on the balance of pro-inflammatory and anti-inflammatory mechanisms and stimuli.

Ideal Value: 3.45

Normal range 2.41 - 7.23:1 M+60 Wm Normal range 2.56 - 7.70:1 MW Normal range 1.94 - 5.82:1 Child

Vitamin K/Arginine - Vascular profile

The term vascular means any anatomical formation, physiological or morbid process which is connected to the blood or lymphatic vessels.

Useful ratio for assessing vascular capacity and efficiency, nitric oxide synthesis, the main vasodilator, to be assessed in relation to blood circulation, correct tissue nutrition, it can provide information on sexual organs and functions, hair structure and health, the systemic and peripheral conditions of microcirculation.

Ideal Value: 1.75

Normal range 1.31 - 3.93:1 M+60 Wm Normal range 1.32 - 3.97:1 MW Normal range 0.86 - 2.59:1 Child

Vitamin E/Tyrosine - Regenerative coefficient

Vitamin E plays an essential role in protecting and repairing the cell membrane from damage caused for example by free radicals. The vitamin has an important role as an antioxidant in preventing the oxidation of polyunsaturated fatty acids, a key event in the development of the peroxidation process. Triggered by the action of free radicals, this event develops through chain reactions which continue the process.

Ideal Value: 1.32

Normal range 0.97 - 2.92:1 M+60 Wm Normal range 0.96 - 2.89:1 MW Normal range 0.71 - 2.13:1 Child

Vitamin B6/Taurine - Hepatobiliary profile

The hepatobiliary profile refers to the liver and the intrahepatic and extrahepatic biliary tracts. The ratio refers to hepatobiliary function and the relationships it has with the activity and functionality of neurobiological systems and tissues and the nervous expression thereof in a psychophysical sense.. Mild hepatobiliary disorders and dysfunctions are often linked to various symptoms, related to intestinal digestive and assimilation functions, and to conditions of general malaise of an undefined nature, frequently identified as "psychosomatic".

Ideal Value: 2.12

Normal range 1.56 - 4.70:1 M+60 Wm Normal range 1.49 - 4.47:1 MW Normal range 1.18 - 3.56:1 Child

Function group sub-unit 3 Hormones/Hormones Tryptophan/Serotonin - Sleep-wake profile

A regular sleep-wake cycle ensures that the biological clock of each person has a positive influence on hormone production, obtaining a sufficient condition of daytime alertness and a satisfying night's rest. It is important that these two phases are in a fairly constant and consistent balance, representing a reliable index of overall good psychophysical health.

Ideal Value: 5.34

Normal range 3.37 - 10.13:1 M+60 Wm Normal range 3.23 - 9.70:1 MW Normal range 4.07 - 12.22:1 Child

Histidine/Noradrenaline - Sensitivity and intolerance index

The ratio refers to the pathogenetic mechanisms of chemical and food sensitivities and intolerances in the context of non-toxic reactions, i.e., not due to reactions from the ingestion of contaminated food or from toxic or poisonous substances, as some are contained in certain fungi.

Ideal Value: 1.52

Normal range 1.00 - 3.00:1 M+60 Wm Normal range 1.09 - 3.28:1 MW Normal range 0.94 - 2.84:1 Child Tyrosine/Dopamine - neurotransmission coefficient

Neurotransmission is the path of steps, consisting of a set of processes and activities, which neurotransmitters follow from the moment of synthesis to the moment of the final effect. In the adrenal gland, tyrosine is converted into levodopa by the enzyme tyrosine hydroxylase-TH. TH is involved in the synthesis of dopamine, noradrenaline and adrenaline as well.

Ideal Value: 1.50

Normal range 1.00 - 3.01:1 M+60 Wm Normal range 0.97 - 2.93:1 MW Normal range 1.03 - 3.10:1 Child

Hormone management index 1 (HMI 1 )

Estrogen/Testosterone

Estrogens are steroid hormones, produced by androgens by enzymatic action, which perform a main function of stimulation and modulation of the genital apparatus and of female sexual behavior; at puberty they promote the development of the genital system and regulate body development. Subsequently they maintain trophism and, together with progesterone, control and condition the physiological succession of menstrual cycles. The regulation of estrogen production during ovulation is controlled by follicle stimulating hormone and luteinizing hormone.

Ideal Value: 4.69

Normal range 2.73 - 8.19 M+60 Wm Normal range 2.57- 7.73 MW Normal range 4.08 - 12.25 Child

Hormone management index 2 (HMI 2) Oxytocin/Progesterone

Oxytocin is a hormone secreted by the neurohypophysis. The main action of oxytocin is to stimulate the contractions of the smooth muscles of the uterus. Progesterone exerts other less important functions as well, always concerning the female genital system, such as modifications of the structure and functional activity of the fallopian tubes and breast. Lastly, during pregnancy progesterone is produced in large quantities by the placenta, and prevents additional ovulation from occurring during the same.

Ideal Value: 15.59

Normal range 9.35 - 28.06 M+60 Wm Normal range 9.35 - 28.06 MW Normal range 12.77 - 38.31 Child

Neuromodulation index 1 (NMi 1 )

Serotonin/Noradrenaline

Neuromodulation is an electrophysiological or chemical process which interacts, in a modifying sense, with the normal sensory neurotransmission activity of the central nervous system. The term neuromodulation additionally means a therapy concept which reversibly intervenes on nerve impulses to change them in terms of stimulus or inhibition according to the needs of the individual, for example in cases of chronic pain, psychiatric disorders, movement disorders, headache, etc.

Ideal Value: 9.5

Normal range 2.88 - 8.64:1 M+60 Wm Normal range 2.73 - 8.19:1 MW Normal range 3.78 - 11.34:1 Child BIBLIOGRAPHY

I. Watts DL, 2003. Trace Elements and Other Essential Nutrients: Clinical Application of Tissue Mineral Analysis. Alexandria: Inter Clinical Laboratories Educational Publications. 2. Watson, G., Nutrition and Your Mind, Bantam Books, NY, 1972.

3. Watson, G., Personality Strength and Psychochemical Energy, Harper and Row, NY, 1979.

4. Wilson LD, 1998. Nutritional Balancing and Hair Mineral Analysis. Prescott: L.D. Wilson Consultants Inc. 5. Eck P., 1990. The American Cranberry. Chapel Hill: Rutgers University Press.

6. Rodale J.l. 1988. The Complete Book of Vitamins and Minerals for Health. Emmaus: Rodale Press.

10. Children's Health and the Environment - A global perspective. A resource guide for the Health Sector, WHO, 2005; American Academy of Pediatrics Committee on Environmental Health. Pediatric Environmental Health, 2nd Ed. Etzel RA, Ed Elk Grove Village, IL: American Academy of Pedriatrics, 2003.

II. Children's Health and the Environment - Mercury, WHO Training Package for the Health Sector, WHO, Jul 2008, pag. 46.

12. Seidel S, Kreutzer R, Smith D, Me Neel S, Gillis G. 2001. Assessment of commercial laboratories performing hair mineral analysis. JAMA. 285: 67-72.

13. J Am Soc Nephrol 18: 2649-2652, 2007. American society of nephrology. Mechanism of Hypokalemia in Magnesium Deficiency. Chou-Long Huang and Elizabeth Kuo.

14. Curr Med Chem. 2004 May;ll(10):1345-59.Proanthocyanidins in health care: current and new trends. Cos P, De Bruyne T, Hermans N, Apers S, Berghe

DV, Vlietinck AJ.

15. Proc Soc Exp Bio! Med. 1999 Dec;222(3):196-204. On the rette of vitamin C and other antioxidants in atherogenesis and vascular dysfunction. Frei B.