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The Effects of cAMP Single Potencies and Mixed Potencies on Acid Phosphatase Activity
Günther Harisch, D.V.M.; Joachim Dittmann, Ph.D.
Reprinted from Biologische Medizin (1999 Feb) 1:4–8.
Abstract
This study investigates whether the effects of cAMP Injeel® and cAMP Injeel® forte in a cell-free system are unique to these preparations or simply equal the additive effects of their single-potency components. When one of the single-potency components of cAMP Injeel® and Injeel® forte was selected as a base potency and the others were added in succession, recorded enzyme activity correlated with the number of potencies added. In all cases, the inhibiting effect of mixed potencies was greater than that of the single potencies, but in no case did the level of inhibition produced by a mixture equal the sum of the effects of its single-potency ingredients.
Introduction
Potency chords are mixed-potency preparations containing equal portions of three potencies of the same substance—a base potency and two additional levels. These potency chords are available commercially under the trade name Injeel®. The “forte” variation includes four rather than three different potencies.¹
The authors confirm the results of the animal experiments reviewed in the article on potency chords in Volume 6/98 of Biologische Medizin (Franke W: Efficacy of Homeopathic Dilutions in the Form of Potency Chords. J Biol Med. 1998;27(6):276–278). The work of the authors on the effects of single and mixed potencies of cAMP on acid phosphatase activity offers additional impressive proof (probability of error 1%; p < 0.01) that potency chords are superior to single potencies in efficacy.
Hartmut Heine, Ph.D.
Materials and Methods
Active Agents
The active substance cAMP was used in the following forms: 6X, 12X, 30X, 200X, cAMP Injeel® (12X/30X/200X), and cAMP Injeel® forte (6X/12X/30X/200X). All preparations were used in liquid form and were supplied in 5 ml ampules by Biologische Heilmittel Heel GmbH, Baden-Baden. (Please note that these cAMP Injeels® are not available commercially.) All cAMP potencies, as well as the Injeel® and its Injeel® forte variation, were produced according to homeopathic principles. The study utilized a blind test format; all preparations were coded prior to use but decoded prior to statistical analysis.
Chemicals
The synthetic enzyme substrate p-nitrophenyl phosphate was supplied by Serva of Heidelberg (Cat. # 30770). All other chemicals used were of the highest available degree of purity.
Enzyme Test System
The enzyme used was acid phosphatase (AP) derived from potatoes (Boehringer, Mannheim; Cat. # 108197). This enzyme model is biologically relevant because acid phosphatase occurs naturally in the lysosomes of human cells. For use in the experimental setups, the enzyme was diluted 1 : 200 with 10 mM of NaAc (pH 5.6).
Incubation Technique
The catalytic activity of AP was measured by determining the amount of p-nitrophenol formed in a microtiter plate assay. 20 µl of the enzyme suspension (out of a total volume of 120 µl) and the cAMP preparation being tested (or water, in the case of the control) were preincubated together at 30 °C. Two series of assays were performed. In one, the preincubation period was 20 minutes; in the other, 40 minutes. After preincubation, each batch of assay components was mixed with 100 µl of the synthetic substrate (5.5 mM p-nitrophenyl phosphate in 0.1 M citrate buffer, pH 5.6) and reincubated at 30 °C. After five minutes, the reaction was stopped by adding 100 µl of 1 N NaOH, and the quantity of enzymatically formed p-nitrophenol was determined using a temperature-controlled microtiter plate reader (ATT C 340, SLT Instruments, Crailsheim) at a wavelength of 405 nm.
The quantity of p-nitrophenol was determined by applying the reference equation p-nitrophenol [nmol × ml⁻¹] = 64.82 × OD 405 nm – 3.373; correlation coefficient 0.998. p-nitrophenol in various concentrations served as the reference substance.
Figure Captions
Fig. 1: Activity of acid phosphatase (AP) in the presence of different cAMP preparations. The graph presents average values (± standard deviation). The line segment ending in circles represents the control. Statistics (n = 48): All experimental mixtures differed significantly from the control (p < 0.01), and 12X differed significantly from all mixtures (p < 0.01). To show the linear relationship among the values more clearly, the y axis begins at 4 instead of at 0, and a line has been drawn connecting the average values for 12X and the Injeel®. The table below the graph shows the composition of the experimental mixtures.
A) Graph (reproduced qualitatively)
(The original plotted mean AP activity [nmol · min⁻¹ · ml⁻¹] ± SD for each setup, y-axis running from 4–10, control marked by ●, and a trend line connecting 12X → Injeel®.)
B) Assay‐Component Table
| Component → | 12X | 12X / 30X | 12X / 30X / 200X | Injeel® (12X/30X/200X) |
|---|---|---|---|---|
| 12X | 25 µl | 25 µl | 25 µl | 75 µl |
| 30X | – | 25 µl | 25 µl | – |
| 200X | – | – | 25 µl | – |
| Injeel® stock* | – | – | – | 25 µl |
| Water | 75 µl | 50 µl | 25 µl | 25 µl |
| Enzyme (AP) | 20 µl | 20 µl | 20 µl | 20 µl |
*In the “Injeel®” column this refers to the mixed‐potency stock (12X / 30X / 200X).
Fig. 2: Activity of acid phosphatase (AP) in the presence of different cAMP preparations. The graph presents average values (± standard deviation). The line segment ending in circles represents the control. Statistics (n = 48): All experimental mixtures differed significantly from the control (p < 0.01), and 30X differed significantly from all mixtures (p < 0.01). To show the linear relationship among the values more clearly, the y axis begins at 4 instead of at 0, and a line has been drawn connecting the average values for 30X and the Injeel®.
A) Graph
(Original mean ± SD plot, y-axis 4–10 nmol·min⁻¹·ml⁻¹, control ●, trend line connecting 30X → Injeel®.)
B) Assay-Component Table
| Component → | 30X | 30X / 200X | 30X / 200X / 12X | Injeel® (12X/30X/200X) |
|---|---|---|---|---|
| 30X | 25 µl | 25 µl | 25 µl | – |
| 200X | – | 25 µl | 25 µl | – |
| 12X | – | – | 25 µl | – |
| Injeel® stock* | – | – | – | 75 µl |
| Water | 75 µl | 50 µl | 25 µl | 25 µl |
| Enzyme (AP) | 20 µl | 20 µl | 20 µl | 20 µl |
Fig. 3: Activity of acid phosphatase (AP) in the presence of different cAMP preparations. The graph presents average values (± standard deviation). The line segment ending in circles represents the control. Statistics (n = 48): All experimental mixtures differed significantly from the control (p < 0.01), and 200X differed significantly from all mixtures (p < 0.01). To show the linear relationship among the values more clearly, the y axis begins at 4 instead of at 0, and a line has been drawn connecting the average values for 200X and the Injeel®. The table below the graph shows the composition of the experimental mixtures.
A) Graph
(Original mean ± SD plot, y-axis 4–10 nmol·min⁻¹·ml⁻¹, control ●, trend line connecting 200X → Injeel®.)
B) Assay-Component Table
| Component → | 200X | 200X / 12X | 200X / 12X / 30X | Injeel® (12X/30X/200X) |
|---|---|---|---|---|
| 200X | 25 µl | 25 µl | 25 µl | – |
| 12X | – | 25 µl | 25 µl | – |
| 30X | – | – | 25 µl | – |
| Injeel® stock* | – | – | – | 75 µl |
| Water | 75 µl | 50 µl | 25 µl | 25 µl |
| Enzyme (AP) | 20 µl | 20 µl | 20 µl | 20 µl |
*In the “Injeel®” column this refers to the mixed-potency stock (12X / 30X / 200X).
| Setup | Composition | Volumes Added |
|---|---|---|
| 1 | 6X | 25 µl cAMP 6X + 75 µl water |
| 2 | 6X / 12X | 25 µl 6X + 25 µl 12X + 50 µl water |
| 3 | 6X / 12X / 30X | 25 µl 6X + 25 µl 12X + 25 µl 30X + 25 µl water |
| 4 | 6X / 12X / 30X / 200X | 25 µl 6X + 25 µl 12X + 25 µl 200X + 25 µl water |
| 5 | Injeel® forte | 100 µl Injeel® forte stock (6X/12X/30X/200X) |
| 6 | Control | 100 µl water |
Fig. 4: Activity of acid phosphatase (AP) in the presence of different cAMP preparations. The graph presents average values (± standard deviation). The line segment ending in circles represents the control. Statistics (n = 48): All experimental mixtures differed significantly from the control (p < 0.01), and 6X and 6X/12X differed significantly from Injeel® forte (p < 0.01). To show the linear relationship among the values more clearly, the y axis begins at 4 instead of at 0, and a line has been drawn connecting the average values for 6X and the Injeel® forte. The table below the graph shows the composition of the experimental mixtures.
A) Graph
(Original mean ± SD plot, y-axis 4–10 nmol·min⁻¹·ml⁻¹, control ●, trend line connecting the series 6X → Injeel® forte.)
B) Assay-Component Table
| Component → | 6X | 6X / 12X | 6X / 12X / 30X | 6X / 12X / 30X / 200X | Injeel® forte (6X/12X/30X/200X) |
|---|---|---|---|---|---|
| 6X | 25 µl | 25 µl | 25 µl | 25 µl | – |
| 12X | – | 25 µl | 25 µl | 25 µl | – |
| 30X | – | – | 25 µl | 25 µl | – |
| 200X | – | – | – | 25 µl | – |
| Injeel® forte stock* | – | – | – | – | 100 µl |
| Water | 75 µl | 50 µl | 25 µl | 25 µl | – |
| Enzyme (AP) | 20 µl | 20 µl | 20 µl | 20 µl | 20 µl |
*In the “Injeel® forte” column, this refers to the mixed-potency stock (6X / 12X / 30X / 200X).
Statistics
The measured values obtained for all setups (activity per volume in nmol × min⁻¹ × ml⁻¹) were subjected to single-factor variance analysis (ANOVA). Subsequently, the Fisher LSD test was performed to directly compare the effect of the control to that of each cAMP preparation or stage in the additive series of cAMP potencies. A probability of error of 1% (p = 0.01) was chosen as the limit of significance.
Results and Discussion
cAMP Injeel®
The cAMP Injeel® used in these experiments is a 1 : 1 : 1 mixture of the single potencies 12X, 30X, and 200X. In numerical terms, adding 75 µl of this mixture to a setup adds 25 µl each of 12X, 30X, and 200X. The design of our experiments took this fact into account, as exemplified by Series 1 (Figure 1):
- Setup 1 (12X) contained 25 µl of cAMP 12X and 75 µl of water.
- Setup 2 (12X/30X) contained 25 µl each of 12X and 30X, plus 50 µl of water.
- Setup 3 (12X/30X/200X) contained 25 µl each of 12X, 30X, and 200X.
- Setup 4 (Injeel®) contained 75 µl of cAMP Injeel® and 25 µl of water.
- Control contained 100 µl of water.
Each setup was mixed with 20 µl of AP suspension and preincubated at 30 °C. After 20 minutes, 100 µl of the substrate solution were added and incubation was continued for five more minutes. The reaction was then stopped and the quantity of enzymatically formed p-nitrophenol was determined through spectrophotometry.
As Figure 1 shows, all cAMP preparations inhibited the catalytic activity of AP. A linear decrease in enzyme activity is apparent in the series 12X → 12X/30X → 12X/30X/200X → Injeel®. A similar tendency was observed both when 30X was chosen as the base potency and 200X and 12X were added one at a time (Figure 2), and when 200X was chosen as the base potency and 12X and 30X were added (Figure 3).
A linear decrease in AP activity was also apparent when the preincubation period was increased from 20 to 40 minutes. Inhibition of AP was more moderate, however, and the recorded values were different (data not shown).
cAMP Injeel® forte
cAMP Injeel® forte is a mixed potency consisting of equal parts of the single potencies 6X, 12X, 30X, and 200X; thus, 100 µl of this mixture contains 25 µl each of 6X, 12X, 30X, and 200X. See the table for the components and test sequence leading to the Injeel® forte variant.
The experiments were conducted in the same way as the Injeel® experiments. As Figure 4 shows, all of these cAMP preparations inhibited AP catalytic activity. A linear decrease is again apparent in the series leading from 6X to the Injeel® forte. The same phenomenon is apparent when 12X, 30X, or 200X is chosen as the base potency (data not shown).
From these results, we can conclude that the effects of cAMP potency chords are not identical to the sum of the effects of the individual potencies they contain. As this example shows, potency chords such as Injeel® or Injeel® forte preparations seem to have qualitatively new and unique effects. Further investigation will be needed in order to determine whether this conclusion also applies to Injeel® and Injeel® forte preparations of other substances.
References
- Wissenschaftliche Abteilung der Firma Biologische Heilmittel Heel GmbH. Ordinatio Antihomotoxica et Materia Medica. Baden-Baden. 1996:13.
- Stryer L. Biochemie. 4. Auflage. Heidelberg: Spektrum. 1996:779–802.
- Harisch G, Dittmann J. Untersuchungen zur Wirkung von Ubichinon-Injeel und Injeel forte mit zellfreien Systemen. Biol Med. 1997;26(3):99–104.
- Harisch G, Dittmann J. Einfluss von cAMP-Potenzen auf die katalytische Aktivität der Sauren Phosphatase. Erste Hinweise für divergierende Wirkungsqualitäten von Einzelpotenzen und Potenzmischungen. Biol Med. 1998;27(5):212–219.
For the authors
Joachim Dittmann, Ph.D.
Institute for Physiological Chemistry
Veterinary College of Hannover
P.O. Box 71 11 80
D-30545 Hannover
Germany