Different biological fluids and the waters in oceans, rivers, lakes, etc., are natural multicomponent water solutions. In order to be analyzed, these multicomponent water solutions need rapid, highly sensitive, specific, and cost-effective IVD assays. Such assays should be similar to those IVDs that are essential for screening and monitoring functional conditions in the human body, general health, and different diseases of large populations of humans, animals, and plants. Such assays should also be able to study the relationships between organisms and the environmental ecosystems of bodies of water.

Any contamination of water in the environment influences other biological organisms.1 This problem is particularly evident with regard to latent types of biologically, chemically, and physically damaging factors. These damaging factors may lead to the development of diseases that are asymptomatic early on, and may not reveal any visible changes in blood and urine samples monitored by traditional laboratory analysis. However, these damaging factors can gradually lead to the development of irreversible and permanent pathological changes in either all afflicted subjects or a group genetically predisposed by genotype or phenotype to these afflicting factors (e.g., diabetes, AIDS, cancer, mad cow disease).2,3

New crystallographic and chemiluminescent IVD assays have been developed that can determine identical types of messaging biomarkers in different biological fluids and ocean waters.1,4-7 With such assays, acquiring fundamental clues about the transfer of information in organism-environment ecosystems is possible. Such IVD assays also open a new approach to screening and monitoring a patient’s general health, diseases of different biological organisms in their early stages, and the effectiveness of treatments. This article will demonstrate the utility and performance of crystallographic and chemiluminescent assays for analyzing different biological fluids and various types of fresh and salt water.

Specificity and Sensitivity

The crystallographic assay’s specificity and sensitivity were examined by adding different organic and inorganic components to a crystallization matrix, and studying native samples of biological fluids and the consequent crystallization of them at certain temperatures, humidity, speed, and time of crystallization in a specially-developed crystallization device. The centrifugation and crystallization of the samples from biological fluids or natural waters required 1.5 hours at room temperature, and the imaging analysis took 1–2 minutes. The device allowed crystallization of numerous samples simultaneously.

In addition, the composition of identical samples was evaluated by high-performance liquid chromatography, gel filtration chromatography, electrophoresis in polyacrylamide gel, mass spectrometry (MS), electron microscopy, diffraction analysis, and paramagnetic resonance (PMR) and nuclear magnetic resonance (NMR) spectroscopy. These highly sensitive techniques usually require very specific processing of biological samples. But by using the crystallographic assay, all components of the sample were crystallized without any specific processing (except for centrifugation of the sample), resulting in no net loss of any relevant and informative physiologically active compounds.

For example, the performance and cost effectiveness of the crystallographic assay are demonstrated by the addition of L-treonine and D,L-treonine to the crystallizing matrix (see Figure 1 top and bottom). The addition of L or D optic isomers to a crystallizing matrix developed a visibly different pattern of crystals: L-treonin crucial and DL-treonin marble patterns. Obtaining such similar information using traditional analytical methods requires expensive and very labor-intensive equipment.

Figure 1. Morphostructure crystal L-threonine (top). Morphostructure crystal D, L-threonine (bottom).

The nature of the crystals was studied by electronograph and diffraction of electrons of the crystals. The analysis of biological fluids and samples of ocean water concluded that their form is hexagonal. The diffraction of electron study showed that the characteristic size of the crystal net is 5A, which in turn shows that the substances that create crystals patterns are not inorganic salts or pure proteins but rather complexes of organic (amorphous) and inorganic (monolithic) low-molecular substances. The study found that the sensitivity of the crystallographic assay for different organic and inorganic additions was 10-8 to 10-16 mol/L and the specificity was 97%.

Analyzing Crystallized Saliva

The crystallization of the saliva from healthy patients and patients with different diseases is shown in Figures 2 and 3. The saliva from bees (A), wasps (B), and other creatures (C, D; see Figure 4), and the bioliquid from healthy and diseased plant leaves (see Figure 5) has shown high reproducibility of crystal structures in normal and pathological samples. Unique changes are found in different biological fluids related to not only the type of biological fluid but also different functional conditions of the biological subject.

For example, patterns of the crystals from normal human saliva produce narrow rod-like crystals with numerous branches at an angle of about 70° relative to the pivotal element (see Figure 2). As a result, the general aspect of normal saliva is a network of oblique lines (p<0.5), although such patterns have small monthly fluctuations related to hormonal cycles. However, the crystals are significantly different from pathological fluids. For example, in human subjects with gastric cancer, the salivary crystals retain their original configuration, but their branching is less pronounced, and the central rods largely account for an increase in the total crystal mass. Its overall incremental increase appears to be threefold to fourfold compared with the crystal mass in normal subjects.

Figure 2. Salivary crystals from a healthy person and from patients with blood cancer, stomach cancer, a malignant gastric ulcer, rectal cancer, gastritis, intestinal cancer, and a gastric ulcer.

The crystal branching in subjects with colon and rectal cancer is even less developed, but the crucial branches are much thicker. As a result, the crystals form isolated aggregates in the shape of multipointed crosses. In gastritis and benign gastric ulcers, the salivary crystals lose their regular shape and have a vague outline in some cases. If the patterns of the crystals in subjects with stage-three gastric cancer and malignant gastric ulcers (an earlier stage gastric cancer) are compared, the patterns are very similar. This example demonstrates that the above-mentioned crystal’s messaging biomarker can determine specific diseases at very early stages.

For example, the ability to determine the level of x-ray disease early on can be demonstrated by the diagnosis of radiation disease in pilots who flew over nuclear reactors (see Figure 3). Pilots were screened and placed into different groups based on the potential effects of radiation disease by using the crystallographic assay. In other words, this process will allow physicians to take essential actions at earlier stages of disease.

Basically, a crystal’s structure depends on specific changes in the saliva’s composition, which reflect metabolic changes at the molecular level in the body. This was proven by highly sensitive traditional analytical technologies. For example, MS and PMR analysis of salivary samples from patients with colon cancer showed the appearance of a range of new, low-molecular-weight substances with MW of 141, 157, 219, and 379 Da. NMR spectres detected the appearance of substances containing group X-CH2-CH2-Y with a chemical shift of 3.6 and 3.7 md. An amino acid analysis showed the appearance of such amino acids as ß-aminosoil acid and y-aminosoil acid. Characteristic patterns of low-molecular-weight proteins in the saliva of patients with rectal cancer include the appearance of new 300-600 and 11,500 Da metabolites, the absence of components with a molecular weight of 9,000 Da, and a marked decrease of neutral proteinase activity.

These findings and the occurrence of the same changes in individual cases provide unambiguous evidence of their specific nature. Being a circulating biological fluid, saliva not only exhibits disturbances of metabolic regulation in various pathological conditions but its changes in composition also participate in the development of a specific biofeedback signal. For example, extensive studies have shown that salivary neutral proteinases are involved in the biosynthesis of selected physiologically active proteins and peptides, triggering and maintaining many biological processes, and inducing adaptive metabolic responses. Their impaired activity may serve as markers for most of the significant changes in the key regulatory mechanisms.6 These findings are also indicative of the changes in biochemical homeostasis associated with the development of the pathological process.

Figure 3. Morphostructure of salivary crystals in different stages of radiation disease.

The crystallographic assay also specifically changed the structure of crystals depending on the presence of particular components in water solutions. This was shown by additional examples studying the ability of the crystallographic assay for rapid and cost-effective quality control of pharmaceuticals. This study demonstrated that each drug developed has significantly different crystal patterns.

The results of research conducted during the past 25 years have demonstrated that if, after a patient’s operation or other treatment, the structure of a salivary crystal is the same as it was before treatment, the patient continued to have the same diseases in the same areas or developed metastasis within 3-6 months. But if a treatment were successful, the crystal of the saliva assumed a normal structure (p<0.05). The crystallographic assay is primarily useful for not only diagnosis but also determining the return of cancer or the possibility of metastasis. By analyzing the structure of the patterns of a messaging crystal biomarker, it is possible to not only determine which diseases or metabolic disorders are present but also monitor the effectiveness of the treatment.

Similar studies with identical samples of biological fluids and different types of microflora (both alive and dead) were conducted with the chemiluminescent assay. The studies showed that the sensitivity of this assay is 10-9 to 10-16 mol/L. The chemiluminescent assay specifically changed the efficacy and kinetics of chemiluminescent reactions. This assay is much more rapid than the crystallographic assay, with the preparation and analysis of one sample taking 3-5 minutes.
An example of early detection of qualitative and quantitative changes in biological fluids (e.g., saliva) is demonstrated by the changes in the efficacy of chemiluminescent reactions during the pathological incubation period. Normally, the level of chemiluminescent efficacy in saliva fluctuates daily within a range of 17–25 units for a period of one year (see Figure 6 top). However, during the development of a pathology (i.e., flu), chemiluminescent efficacy changes and increases up to 50 units and more, which happens 6-7 days prior to the appearance of any clinical symptoms of the disease. (See Figure 6 top.)

Figure 4. Salivary crystals from a bee, a wasp, a hornet, and a snake.

Analyzing Ocean Waters

The crystallographic and chemiluminescent assays were also used to analyze ocean waters. The presence and distribution of crystal messaging biomarkers and the efficacy and kinetics of chemiluminescent reactions in normal environments (see Figure 7) as well as during disturbances of the water environments by various interfering factors (see Figures 8, 9, and 10) were studied.1 The efficacy and kinetics of the chemiluminescent assay and the patterns of the crystals from the crystallographic assay were registered at different depths and distances from specific sources of interference.

The study was conducted in the Mediterranean Sea, Black Sea, Sea of Japan, and in different regions of the Pacific, Indian, and Atlantic Oceans. A study of the background characteristics of different regions of ocean waters at depths of up to 200 meters showed that the registered parameters of the crystallographic and chemiluminescent assays were not absolutely consistent with profiles of temperature, density, or salinity. The assays also demonstrated certain characteristics depending on the specific regions of the oceans, seas, and rivers, and the direction of currents (see Figure 7 A, B, C).

Nonetheless, the background study showed both assays performed within a specific range in each water region. And despite some changes caused by organic-diluted substances and accumulations of bio and phytoplankton, the assays were still within a range consistent with daily and seasonal fluctuations (except at junctions of rivers with oceans). In the water samples from these areas, depending on the type of contaminations, certain crystal structural changes were detected at different locations in sea water at a distance of 25–50 miles offshore (see Figure 10 A, B). The passage of ships or large mammals (e.g., whales) did not cause significant fluctuations in the background parameters of the crystallographic and chemiluminescent assays.

Figure 5. Patterns of crystals from the leaves of a healthy cucumber plant. Patterns of crystals from the leaves of a diseased plant.

The study of the background parameters demonstrated that crystal messaging biomarkers are permanently present in ocean waters (see Figures 7, 9). However, during the appearance of specific underwater factors interfering with the water environment (e.g., detonations, diesel and nuclear submarines, chemical contamination, etc.), the quantity and patterns of crystals, as well as the kinetics and efficacy of the chemiluminescent reactions of the water samples, were significantly changed. Moreover, different types of underwater interfering factors (e.g., nuclear or diesel submarines, explosions) caused significantly different crystal structures (see Figures 8 and 9).

The activities and reactions of the crystallographic and chemiluminescent assays were completely opposite at different distances from an interference (see Figure 9). For example, the efficacy of chemiluminescent reactions started to decrease slowly from the background index at a distance of 159 miles from a nuclear submarine in motion, and at 1–3 miles from a submarine in motion dropped to 0. These changes not only occurred in the submarine’s wake but also were registered in front of it, like a predictor from the moving submarine. An analysis of the changes in the chemiluminescent assays can determine not only the presence but also the exact location of the subject. Possibly, the changes in the ocean spread out in all directions that could register the assays within 159 miles from the submarine, or through biophysics and sound channels in front of it.

Meanwhile, the reactions of the crystallographic assays showed an increase in the amount, size, structure, and specificity of the crystals while approaching a specific interfering source. The maximum specific size and quantity were registered at a distance of 1-6 miles and at all depths up to 200 meters (see Figure 9). In addition, the analysis of river waters with the crystallographic assay demonstrated that specific patterns of crystals can trace the movements and distribution of specific controlled components, and can detect the presence and location of these substances. A computerized graphic analysis of a river-sea junction illustrates such possibilities (see Figure 10).

Figure 6. Representative analysis of the variation of intensity of a chemiluminescent assay for a healthy person during a one year period (top). Representative example of the variation of intensity of chemiluminescent measurements for the saliva of a patient with influenza. Clinical symptoms appear only at day six (bottom).

Discussion

It is well known that specific biochemical metabolic changes at a molecular level develop in the body significantly earlier than functional symptoms of a disease become visible. However, routine blood and urine tests and physical detection methods are not always effective at detecting diseases or biochemical changes at early stages, such as cancer, prediabetes, mad cow disease, etc.5-7

This is particularly important in connection with the increase in bioterrorist activities and the lack of efficiency in traditional systems for biological control of latent damaging factors and asymptomatic diseases. The solution for these problems should be permanent metabolic and environmental controls in organism-environment ecosystems.

For the control of functional conditions in humans and animals, it is more convenient to use saliva. Saliva samples are more easily collected and handled than urine and blood, and allow increases in testing frequency and precise control of metabolism.7 Saliva tests present great opportunities for noninvasive, painless, and cost-effective tools for diagnosing and monitoring routine and asymptomatic diseases. The presence of specific changes in the biomarkers can detect the signs of routine or asymptomatic diseases at early stages (see Figures 2, 3, and 6).6

An enormous amount of research has been dedicated to messaging biomarkers, especially RNA. All of the research established that messaging RNA was permanently present in the saliva and different biological fluids of humans, animals, plants, fish, etc. This messaging biomarker can be purified and crystallized by special techniques on a special crystallization matrix.8 The RNA crystal patterns provided specific and significant information about not only the presence of diseases but also functional conditions of an organism. However, this technique can crystallize only purified peptides or RNA. This technique requires a lot of time for purification and preparation of the samples and an expensive x-ray crystallography analysis (approximately $4000 per analysis) that are not appropriate for mass screening and monitoring out in the field.

Figure 7. Background patterns of crystals and the chemiluminescent efficacy in different depths and distances in ocean waters: Pacific Ocean, Indian Ocean, and Mediterranean Sea.

In order to analyze multicomponent water solutions, pharmaceuticals, and other products with the crystallographic assay, a universal crystallization matrix is needed. One such matrix is mucin. The mucin matrix has been used for crystallizing different biological fluids and natural waters. Mucin is also permanently present in all biological fluids and the biological systems of ocean waters. In addition, mucin is an essential component that participates in biochemical and immunological reactions and in transmitting and regulating components in biological organisms.

Numerous scientific research reports have recognized that messaging biomarkers, especially RNA-DNA, are very sensitive to electromagnetic, chemical, and radio-modulated light and sound signals. These factors can not only change physiological parameters of biological organisms but also affect cells by damaging their membranes and DNA.

The cells in biological organisms and biosystems of natural waters use for transmission and communication not only biochemical or nervous signals but also the above-mentioned physics transmitters for specific molecular biofeedback through biophysics channels. These are essential for maintaining homeostasis in biological populations and the bioenvironments in natural waters. In this situation, it becomes more understandable why in biofluids such as ocean waters that permanently contain different types of bio and phytoplankton, this messaging and transmitter system functions even at great distances. It is possible that in ocean waters and biological fluids of humans, animals, plants, and other biological organisms, there are certain identical or very similar transmitters of information. All of these messaging components are present permanently in the normal background with a defined quantity and quality. However, during the development of any pathology or during the appearance of interfering factors in the water environment, they immediately and specifically change the level of quantity and quality of biochemical components, which is reflected in the patterns of crystals and the efficacy and kinetics of chemiluminescent reactions.

Figure 8. The patterns of crystals and the efficacy of the chemiluminescent reaction in different depths and distances and under the influence of specific underwater disturbances: Pacific Ocean, Atlantic Ocean, and Mediterranean Sea.

Conclusion

To summarize, this article made the following observations:

The results of research conducted in different clinical and agriculture studies, quality control of pharmaceutical products, and environment control concluded that new crystallographic and chemiluminescent IVD assays can be used as simple, sensitive, informative, rapid, and cost-effective assays.

It is quite possible that in world ocean waters and different biological fluids, identical or very similar transmitters of information or messaging biomarkers are present in a biofeedback regulation system that maintains homeostasis and balance in organism-environment ecosystems.

Saliva samples are more easily collected than urine and blood, and allow increases in testing frequency and precise monitoring of metabolism in large populations of humans and animals.

The nature and function of crystallographic messaging biomarkers and specific biophysics channels should be studied in more detail. The knowledge of their structures and functional abilities in transferring information may open a whole new approach to controlling and studying the relationship between environment and biological organisms, and solving a range of applied and fundamental problems in the hydro and biospheres.

References

1. SV Kharchenko, GA Korneeva, and AAVetrov, “Organism-Environment Ecosystem State Assessment According to Chemoluminiscense Assay Parameters,” Izvestiya Academy of Sciences, UDK 551.46 (1991): 299-302.

Figure 9. The relationship between the efficacy of the chemiluminescent signal and the concentration of specific patterns of crystals at different distances and depths from an underwater object.

2. WHO Neil, et al., “Distribution of NAT2 Phenotype in HIV/AIDS,” International Conference on AIDS 11 (1996): 451.

3. RD Lobert, et al., “The Lethal Phenotype of Cancer: The Molecular Basis of Death Due to Malignancy,” CA: A Cancer Journal for Clinicians 57: 225-241.

4. SV Kharchenko, GA Korneeva, and AAVetrov, “Saliva Crystals: the Nature and Properties,” Izvestiya Academy of Sciences, UDK 591.1 (1988): 450–454.

5. SV Kharchenko, GA Korneeva, and AA Vetrov, “Some Changes of Rat Saliva Composition During Development of the Cancer of Mammary Gland,” Izvestiya Academy of Nauk USSR 3 (1987): 124-30.

6. SV Kharchenko, GA Korneeva, and AA Vetrov, “Cancer-Associated Composition Changes in Human Saliva,” Izvestiya Academy of Nauk USSR 4 (1989): 524-30.

7. SV Kharchenko and N Flook, “Detecting Metabolic Changes in Diabetes Mellitus,” IVD Technology 14, no. 4 (2008): 49-57.

8. SR Holbrook, EL Holbrook, and HE Walukiewich, “Crystallization of RNA,” Cellular and Molecular Life Sciences 58, no. 2 (2001): 234-343.

Figure 10. Distribution of different substances that developed crystal patterns type A and type B at a river delta–sea junction.

Sergei V. Khartchenko, MD, PhD, is president and chief executive officer at NewDiaTech Diagnostics Technology Corp.
(Victoria, BC, Canada). He can be reached at newdiatech@shaw.ca.