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  Rhythmic Phenomena of Potentized Substances 2
  

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By: Thomas C. Carmine
Rhythmic Phenomena - 2.doc

(Original title: Rhythmusphaenomene potenzierter Substanzen 2 - Bestimmung der H^Oy-Pro-duktion von Neuroblastomzellen nach Stimulierung mit Hochpotenzen des Zytotoxins TNRri-pha durch Chenioluminiszenz - Ein moegliches System zum Wirksamkeitsnachweis homoeo-pathischer Hochpotenzen. Der Merkurstab 1996; 49:282-9. English by A. R Meuss, FIL, MTA.)

Chemiluminescence Assay of H2O2 Production by Neuroblastoma Cells Stimulated with High Potencies of the Cytotoxin TNFalpha -A Potential System for Demonstrating the Efficacy of Substances in High Potency

Abstract
The effect of high potencies of the cytokine TNFalpha on H2O2 production by SK-N-SH neuroblastoma cells is described. An extremely sensitive amplified chemiluminescence method (ECL) showed marked, reproducible (n = 10) effects of 'TNFalpha on H2O2 production (p < 0.0025, Student t test). The dose-effect relation was studied over a range from 200 ng/ml of TNfalpha to the lOOx potency. Interpolation produced a sinusoid curve (phase interval 5-6) that appeared superimposed on a sinusoid polynomic regression curve of the 10th order (phase interval ~ 35). The results shown in this paper strongly suggest that the chemiluminescence method used is well suited to demonstrate and further investigate the effect of high potencies on biologic systems.

1. Introduction
Many tumor cell lines produce considerable quantities of the reactive oxygen metabolite hydrogen peroxide H2O2.(1) Like other reactive oxygen compounds, this molecule is presumably closely connected with the malignity and growth patterns of tumors.(1,2) A paper published in 1994 described a method of chemiluninescence (The term chemiluminescence was first used by the German chemist, Eilhard Wiedemann in 1888 with reference to "the luminescence seen with chemical processes"(4) if greater in intensity than the temperature radiation.(5) The phenomenon was first mentioned in the present age in connection with the "Bologna stone" discovered by the cobbler and alchemist, Vincenco Cascariolo, in 1603. The stone consisted mainly of barium sulfate and was able to absorb sunlight, emitting it again in the dark.(4)) that permits sensitive assay of H2O2 in supernatants from adherent SK-N-SH neuroblastoma cells. The method based on chemical enhancement of the classic and probably most widely used chemiluminescence reaction, which is the oxidation of luminol







Fig. 1. Simplified representation of luminol oxidation. The current theory is that a number of high-energy excited electronic states of the luminol molecule produced in the course of the reaction are immediately oxidized further, reducing their energy level,(6) with the difference in energy given off as light. The amount of light detected is proportional to the H2O2 concentration if peroxidase and luminol concentrations are constant.


(aminophthalic acid hydrazide) in the presence of H2O2 and peroxidase, which is shown in simplified form in Fig. 1.

An interesting aspect of H2O2 determinations with SK-N-SH neuroblastoma cells was that H2O2 production rose considerably on addition of unphysiologically high concentrations (100 ng/ml) of the cytokine tumor necrosis factor alpha (TNFalpha), and the effect was reversed after several 1:10 dilution stages of the TNFalpha solution, so that the H2O2 concentration was reduced (at a concentration of 0.1 ng/ml). Remarkably, high potencies of TNTalpha (up to the lOOx) had a powerful effect on H2O2 production by SK-N-SH neuroblastoma cells, as will be shown below.

TNfalpha is in vivo mainly secreted by activated macrophages and may produce a number of effects, e.g. tumor necrosis, pyrexia, shock, cachexia or activation of other immune cells.(7) The effects of TNFalpha on in vitro growth of neuroblastoma cell lines depend on the concentration, as does H2O2 release, and probably vary between cell lines. Though often toxic in higher concentrations, TNFalpha was presented as an autocrine growth factor for neuroblastomas by a group of French workers.(8)

The results presented below suggest that it may be possible to extend conventional tumor treatment on this basis. They may also offer new and deeper insights into the potentization process.

2 Method
2.1 Cell culture
SK-N-SH cells (~ 100,000/well) were placed in 48-well plates between 66 and 24 hours before starting the experiment. The cells normally need about 6 hours to become attached to the base of the well. A longer period was allowed to ensure dense population of the base. The plates were checked under







Fig. 2. Dose-effect relationship between TNFafpha and H2O2 production by SK-N-SH cells. Each dot represents the mean for a double determination. With one exception the results always came close together. Individual means per potentizing stage are connected by a finely dotted line. The solid line represents the regression function of the 10th order, the broken line the 100% level.


a microscope before the medium was drawn off and the cells washed twice with 500 mcl of phosphate-buffered saline (PBS + calcium + magnesium, Dulbecco type, from Seromed, Germany). 200 mcl of incubation buffer consisting of 9 parts of PBS+Ca+Mg+5 mM glucose and 1 part of sterile, pyrogen-free water were added to fee cells. The water was either the end point of a TNFaIpha potentization series (in pyrogen-free, sterile water) or it was "pure" water succussed with PBS for the controls. After 60 minutes' incubation, the H2O2 concentration of the buffer was determined. The cells adhered to the base throughout the incubation period. They were removed with trypsin after H2O2 determination and counted in a Neubauer chamber.

2.2 H2O2 determination with chemiluminescence
This was done by the method given by Carmine.(3)

2.3 Potentization ofTNFalpha
Immediately following delivery of recombinant human tumor necrosis factor alpha (specific activity > 108 U/mg, Boehringer, Mannheim) in 10 ug/ml concentration, 20 mcl aliquots were stored at –20 degrees C. Each aliquot was gently thawed on ice before adding 1980 mcl of pyrogen-free, sterile (ultra-filtered) water (Ampuwa). This represents a 1:100 dilution or a TNValpha concentration of 100 ng/ml (= Ix). The resulting solution was used for decimal potentization,








Fig. 3. H2O2 production by SK-N-SH neuroblastoma cells in the presence of TNFafpha and controls. Each dot represents the mean result of 10 separate determinations, details of which are given in Table 1. The bars represent the means of the ten determinations, the error bars the standard deviations. A bilateral Student t test gave a significant difference (p < 0.0025).



at every stage pipetting 100 mcl of the preceding solution into 900 mcl of pyrogen-free water in a 1.5 ml Eppendorf cap. The cap was succussed strongly and rhythmically 10 tunes from the wrist (allowing for its spring). This was done well away from any more powerful electromagnetic fields, e.g. those produced by cassette recorders or electrical shaking machines.

Shortly before the cells were first washed, the incubation butter was produced, i.e. the mixture of 9 parts of PBS+Ca+Mg+5 mM glucose and 1 part of either TNFalpha potentized in water (see also under Cell culture) or "pure" water for the controls. This was also the final dilution stage. The incubation buffer - this refers to both TNFalpha potencies and control buffer -was once more vigorously shaken immediately before adding it to the cells.

3 Results
3.1 Dose-effect relation between TNFalpha and H2O2 production by SK-N-SH cells Figure 1 summarizes the effects of TNFalpha in doses ranging from the 1x to the 100x on H2O2 production by SK-N-SH neuroblastoma cells. The whole experiment was done on three days in one week. The 6-channel chemiluminescence analyzer allows a maximum of 3 double-determinations to be made concurrently (2 TNFalpha potencies and 1 control), so that 50 6-channel determinations were needed (~ 17/day) to assess the 100 potency stages. Testing








Fig. 4. H2O2 production by SK-N-SH neuroblastoma cells in the presence of TNFalpha, potentized water and unpotentized water (= controls), each in the 30x, 70x and 100x. Means of two (in the case of the 100x four) separate determinations, with standard deviations. Inset: Percentage deviation from control for potentized water and TNFalpha H2O2 (pml/104 Zellen) = H202 (pml/104 cells)


the calibration at the beginning of each day when determinations were done showed no differences in intensity between channels. To minimize systematic error, the sequence of samples in the 48-well plates was changed, with sample positions varied by rotation from one determination to the next. To relativize fluctuations between individual determinations, H2O2 production was given as the percentage deviation from the controls relating to the particular determination.

TNFalpha concentrations are entered as logarithms in Figure 2. Interpolation produces a markedly sinusoid curve (finely dotted line), especially in the range above the 30x, the mean phase interval being 5-6 potentizing stages. Polynomic regression analysis (10th order) also gave a sinusoid curve (solid line), its amplitude increasing with potentization. One might also say that numerous small waves are carried on a large basic wave, organized in a way similar to ocean waves, with many tiny wave babies romping about on a large wave coming to the shore, the same on the small scale as on the large.

3.2 Results with TNFalpha 100x
Figure 3 shows the results of ten separate determinations of H2O2 production by SK-N-SH neuroblastoma cells with and without TNFalpha 100x






treatment. The determinations - generally double or triple - were made on 5 separate days. The graph shows all the experiments done; none were excluded. H2O2 production is shown in 10 to the 4th cells/h, i.e. in absolute figures. A bilateral Student t test gave a highly significant difference (p < 0.0025) between the controls and TNFalpha treated cells. Table 1 gives a detailed summary of individual experiments.

3.3 Effect ofpotentized water on H2O2 production by SK-N-SH nenroblastoma cells
Both pure, pyrogen-free sterile water and TNFalpha (100 ng/ml) were poten-tized to (pre)stages 29x, 69x and 99x, using the method given under 2.3, and stored over night protected from light. The next day the solutions were used to produce incubation buffers to examine the effect on H2O2 production by SK-N-SH neuroblastoma cells by the method given under 2.1-2.3.

The results are shown in Figure 4. Potentized water increased H2O2 production both in the 70x and the lOOx, with the effect distinctly stronger than for the comparable TNFalpha potency in the case of the 70x, and distinctly weaker for the lOOx. This strongly suggests that potentization of the diluent has an effect, which, however, only appears to be modified in the characteristic way by TNFalpha.

3.4 Effect of high potencies on the chemiluminescence analyzer system
Different high potencies were regularly added to a "cell-free" system containing defined amounts of commercially available H2O2. No indication was seen of an effect on the analyzer system (data not shown). It may thus be assumed that the results reflect only the effect of the high potency on the neuroblastoma cells, i.e. on the biological system.

4 Conclusion
The results strongly suggest that the highly sensitive chemiluminescence system (ECL) used in these experiments can detect and quantify the actions of high potencies on a cellular system (neuroblastoma cells in the present case).

Thomas C. Carmine
Hartmeyerstr. 2/317
D-720076 Tuebingen
Germany

References
1 Szatrowski TP, Nathan CF. Production of large amounts of H2O2 by human tumor cells. Cancer Res 1991; 51: 794-8.
2 Cerutti PA. Prooxidant states and tumor promotion. Science 1985; 227:375-81.
3 Carmine TC et al. Measurement of endogenous and TNFalpha-mediated H2O2) production in supematants of SK-N-SH neuroblastoma cells with an enhanced chemiluminescence assay. / Biolumin Chemilumin 1994; 9: 267-72.
4 Campbell AK. Chemiluminescence. Chichester: Ellis Horwood 1988.
5 Brolin S, Wettermark G. Bioluminescence analysis. 1. Aufl. Weinheim: VCH 1992.
6 Merenyi G et al. Luminol chemiluminescence: Chemistry, Excitation, Emitter. / Biolumin Chemilumin 1990; 5:53-6.
7 Klein J. Immunology. Oxford: Blackwell Scientific Publications 1990.
8 Goillot et al. TNF as an autocrine growth factor for neuroblastoma. Cancer Res 1992; 52: 3419-200.

Additional bibliography
Davenas E, Benveniste J et al. Human basophil degranulation triggered by very dilute antiserum
against IgE. Nature 1988; 333:816-7. Hirst SJ et al. Human basophil degranulation is not triggered by very dilute antiserum against
human IgE. Nature 1993; 366:525-7.
Bischof M. Biophotonen - Das Licht in unseren Zeilen. Frankfurt a.M.: Zweitausendundeins 1995. Schmid F. Medikamentoese Therapie auf dem Pruefstand. Allopathic - Phytotherapie -
Homoeopathic. Lecture in Marburg on 20 March 1993. Brodeur GM et al. International criteria for diagnosis, staging and response to treatment in
patients with neuroblastoma. / Clin Oncol 1988; 6:1874-81. Evans AE. Natural history of neuroblastoma. In Advances in Neuroblastoma Research. Evans EA
ed. New York: Raven Press 1980.
The results presented in this paper and their discussion have been published in Allg Hom Ztg 1996;241.






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