Category: Publications

DR. MED. W. THIEL

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I. Introduction
One of the most important functions of the skin is to protect the organism against noxae and to guarantee regulated exchange of substances between the organism and its environment. If medicaments have to be brought into the organism by the percutaneous route, then this physiological protective barrier has to be overcome.

One method of transporting active substances through the skin into deeper lying tissue or to increase the extent of normal substance diffusion is direct current therapy. Transcutaneous ion transport, known as ionophoresis, is facilitated by this. Nevertheless, a prerequisite for this is that the pharmacologically active component of the dermatic is present in an electrically charged condition. According to whether the active substance is positively or negatively charged, the medicament is applied beneath the anode or cathode.

More recent studies⁴ ⁵ demonstrate that when ionophoresis is compared with diffusion, increased substance transport takes place only in the horny layer of the horrifying squamous cell layer. Since it is just this horny layer which represents the most effective barrier against diffusion of active substances, greater effectiveness can be achieved here by ionophoresis.

However, the active substances are spread within tissue layers lying below mainly by diffusion along the concentration gradient³.

With regard to the therapeutic effectiveness of this method, a pilot study³ after iontophoretic administration of a non-steroidal antirheumatic agent, humeral epicondylopathy being indicated, revealed a significant decrease in average pain intensity in the case of tenderness and total pain sensation, but further assessment features showed only a slight improvement. However, because of the lack of a comparative group, separation of the medicinal from the physical therapy effect was not possible.

In the case of ointments or gel preparations which contain only one medicament with positive or negative charge, substance transport of greater extent with the aid of direct current is evident because of conformity with natural physical laws¹. The therapeutic effectiveness of a combination treatment of direct current/medicament is also documented empirically elsewhere².

In the case of pharmaceutical mixtures, as are present in phytotherapeutic extracts and homeopathic preparations, it is not known as a rule whether the effective constituents are present in ionized form and if so, what charge they have. In addition there is the fact that it must be assumed in the case of these medicaments that several constituents which can also have different electrical charges are therapeutically effective.

The user can change the current direction at the iontophoresis unit in the course of therapy in order to be able to let both the positively and the negatively charged constituents through the skin into the tissue. Nevertheless, electrically neutral substances cannot be transported more quickly into foreign tissue by this method.

The objective of this investigation was to ascertain whether the combination of a Traumeel ointment therapy with iontophoresis has a better therapeutic result, i.e., faster restoration of health, than application of ointment alone. The therapeutic effectiveness of Traumeel ointment in distortions has been proven without doubt in the course of a double blind study⁶. Taking this effectiveness as proven, the therapeutic comparison of Traumeel ointment versus Traumeel ointment/direct current therapy was sufficient for us in this study without inclusion of a placebo.

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II. Patients and methodology

A total of 50 patients with medium degree injuries were included in the study. All had the same type injury of external ligamentous overstretching in the sense of supination distortion trauma. 25 patients were treated exclusively with Traumeel ointment. The other 25 patients were subjected in addition to direct current therapy.

After the injury, immobilization in a Traumeel ointment dressing was provided for two days for all patients. No further dressings were applied after this.

One group of patients received following this a physiotherapy every 2 days in the shape of iontophoresis with a current intensity of 3 mA for a period of 10 minutes per session. The ointment was applied alternatingly for each patient both on the cathode and on the anode. Cold packs were applied in each case following this treatment. Traumeel ointment for daily self-application was prescribed to the patients of the other group. The patients were examined on the day of injury as well as on the 1st, 3rd, 5th, and 7th day. Assessment criteria were pain at rest, under pressure, and in motion, and were documented by means of a 4 point scale (0 = without, 1 = little, 2 = medium, 3 = severe). Furthermore, the circumference was measured with the aid of a tape measure over the narrowest point, the ankle, and the metatarsus. The mobility of the ankle joint was measured according to the neutral zero method. Circumference and mobility were compared with the healthy side in each case and the change in difference was evaluated. Since the patients were competitive athletes, the period up to the first day of return to athletic training was taken as the criterion for assessing the therapy.

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III. Results

1. Return to training
If the two groups are compared with one another with regard to the first day of return to training, it results that more patients in the group treated solely with Traumeel ointment returned to training at an earlier point in time.

Fig. 1: Return to Training

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2. Pain at rest
Fig. 2: Course of the average pain at rest values

The intensity of the pain at rest was documented in the shape of point values on the 1st, 3rd, 5th, and 7th day of treatment.

The starting value of the two groups were different (with iontophoresis 1.9; without iontophoresis 0.8), so that a comparison of the course with regard to this feature could be made only conditionally. However, it can easily be seen from Figure 2 that both starting values decreased over the 7 days continuously and in a comparable ratio. In the group treated only with ointment, the value of 0.1 was reached for pain at rest within 3 days because of the low starting value; in the iontophoresis group, this value was reached only on the 7th day. (However, the higher starting values existed here.)

Since various patients did not appear at some appointments for “nonmedical reasons”, the starting values of these dropping out patients were analyzed separately. Since these were not extreme values, the average trend values of the assessment features were not distorted by the lack of these data.

3. Pain on pressure

Fig. 3: Course of the average pain on pressure values

The starting values in both groups were comparable with regard to this feature (iontophoresis group on average 2.2; Traumeel group on average 1.9). The intensity of pain on pressure also decreased here continuously as can be seen in Figure 3 from 1.9 points to 0.3 points. In the iontophoresis group, the average pain intensity increased at the beginning (from 2.2 to 2.5) and then decreased continuously but significantly slower than in the Traumeel group. On the 7th day of observation, an average residual pain on pressure of 1.1 was still present.

4. Pain on movement
Fig. 4: Course of the average pain on movement values

The starting values of both groups were also comparable in the case of this feature (iontophoresis group on average 2.2; Traumeel group on average 1.9). The intensity of pain on movement also decreased here continuously as can be seen in Figure 4 from 2.0 points to 0.3 points.

5. Mobility
The mobility of the healthy and injured joint was measured according to the neutral zero method and the difference between the two values was formed.

In the Traumeel group, this difference decreased to the value of 3.8° on the 7th day, a negligible value, which lies in the range of the error limits. In the iontophoresis group, a residual angle difference of 10.7° remained on the 7th day after initial deterioration (Fig. 5).

Fig. 5: Course of the average extent of mobility
(Difference between injured and healthy joint)

6. Circumferential measurements
The data of the circumferential measurements are only conditionally suitable for interpretation (see discussion), since the spread (see table 1) and the value range are too large.

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IV. Discussion
In orthopedic traumatology, there is a trend towards treating the consequences of injury with antiphlogistic ointments and to make therapeutic use of the stimulating effect of direct current treatment (galvanization). The ionization of the active substance is considered to be secondary.

As can be seen from the above tables, an increase in swelling in the ankle joint region was noticed in the control examinations in the iontophoresis group. This is attributable to a condition of irritation caused by electrotherapy as well as to sacculation of the hematoma of the distal joints of the metatarsus.

On critical comparison of the two forms of application of iontophoresis and simple treatment with ointments after inital elastotape dressing, the latter musgt be given preference because of the more rapid incidence of effect, in particular for competitive athletes. Exclusive ointment application has also the advantage of being free of irritation. Side effects were not observed in any patient.

On the other hand, iontophoresis has proven itself for patients who cannot tolerate adhesive dressings for several days (lacing effect in varicosis) or who themselves cannot rub in ointments for certain reasons (older people, disabled etc.). Application of ointment alone, i.e. without initial tape dressings, should be performed only for simple bagatelle injuries which require no specific medical supervision.

Literature

1. Pratzel, H., Die Iontophorese, Med.-Info Nr. 1 (Transcutan GmbH)
2. Magyarosy, J., Ernst, E., Marr, N., Schmolzl, Ch., …
3. Schops, P., Seichert, N., Erdl, R., Siebert, W., Pratzel, H., Pilotstudie zur klinischen Wirksamkeit einer definierten Iontophorese mit Indometacin bei Epicondylopathia humeria, Z. Phys. Med. Baln. Klin. 15, 395–399 (1986)
4. Pratzel, H., Grundlagen des perkutanen Stofftransports in der Pharmako-Physio-Therapie und Balneotherapie, Dissertation 1985 (quoted in 3)
5. Pratzel, H., Machens, R., Dittrich, P., Iontophorese zur forcierten Hautresorption von Indometacin und Salicylsaure, Z. Rheumatol. 104, 40, 748 (1986) (quoted in 3)
6. Zell, et al., publication in preparation

Address of the author:
Dr. med. Werner Thiel
Orthopedic physician
Kirchstraße 9
D‑6635 Bous
Germany

Günther Harisch, D.V.M.; Joachim Dittmann, Ph.D.

Reprinted from Biologische Medizin (1999 Feb) 1:4–8.

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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®.)

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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®.)

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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.)

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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

1. Wissenschaftliche Abteilung der Firma Biologische Heilmittel Heel GmbH. Ordinatio Antihomotoxica et Materia Medica. Baden-Baden. 1996:13.
2. Stryer L. Biochemie. 4. Auflage. Heidelberg: Spektrum. 1996:779–802.
3. Harisch G, Dittmann J. Untersuchungen zur Wirkung von Ubichinon-Injeel und Injeel forte mit zellfreien Systemen. Biol Med. 1997;26(3):99–104.
4. 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

J. Kersschot, M.D.
Belgium

BioMedical Therapy Magazine Symposium
At the Royal Society of Medicine, London
10. 5. 1997

A. Introduction

Although the use of corticosteroids is accepted as a standard technique in the treatment of several inflammatory diseases, we must recognize that specific injection techniques of anti-inflammatory products are gaining more and more interest. Most physicians, however, tend to regard these techniques as marginal phenomenons. The importance of these injections in the treatment of so-called inflammatory diseases is relatively unexplored and promises to be a fertile area for further investigation.
Since most physicians have never been trained in these techniques, they are anything but expert in this field. I think that every therapist should at least know about the existence of this strategy, even if he or she will not use injections in his or her own practice.
So, I will introduce to you today the therapeutical strategies that focus on injections of non-steroid products. Both chemotherapeutic and biotherapeutic products will be discussed, and several techniques of administrating them will be explained. The clinical cases will try to illustrate my strategies.
I have more than ten years of experience with these techniques in my private practice as a general practitioner in Belgium, and I have noticed that there is a very broad spectrum of medical problems that can be managed with this injection strategy. Today, however, I will only discuss two examples of alternatives for cortisone: the treatment of musculoskeletal pain and the treatment of asthma.
Still, I do not make any claims about the injections described in this lecture, involving the prevention or cure of any disease. Maybe the effects that I have noticed in my private practice are no more than a sophisticated placebo. I cannot be sure that what I am doing is best for the patient unless this practice has been rigorously tested; to check the items that I suggest, large-scale clinical studies are necessary. This lecture can be regarded as an invitation to do so in the near future.

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B. Injection of biotherapeutics

B.1. Injection of biotherapeutics is not a very strategic novelty

Those familiar with biotherapeutics for injection know that many ampoules exist since several decades, especially in Germany. Especially Heel, Pflüger, Steigerwald, Dolisos, Cosmochema, Fides and Hevert are famous for their ampoule preparations. Thousands of them are injected every day, and clinical studies check their efficiency and control their possible side-effects (ref. 20, 21, 28, 29). A wide selection of homeopathic and phytotherapeutic preparations has been on the market in injection form for many years now, and they have been employed in daily medical practice with good success, especially by general practitioners, rheumatologists and orthopaedic doctors. Many authors have already mentioned the use of biotherapeutic ampoules for injection: Bianchi (ref. 3), Claussen (ref. 13), Coeugniet, de La Fuye (ref. 31), Diamond (ref. 44, 45), Fischer (ref. 36), Frase, Gellman (ref. 19), Graf von Ingelheim (ref. 27), Geyer (ref. 24), John, Kleinscholt, Küstermann, Lannugier-Bolling, Meltelmann (ref. 28, 29), Müller, Pollmann, Potrafki (ref. 21), Preusser, Reckeweg (ref. 14), Riley (ref. 16), Risch, Schmid (ref. 17), Subotnick (ref. 43, 48), Thiel (ref. 20), Timmermann (ref. 10), Vorstoffel (ref. 33), Wachter, Werthmann, Weiser (ref. 28), Zenner (ref. 29) and many others (see ref. 1: bibliography).
Since there was no specific name for all the methods that use biotherapeutics for injection on specific spots, I introduced in Belgium the term ‘biopuncture’ (ref. 1, 2, 5), in order to distinguish the use of homeotherapy according to de La Fuye (ref. 31), neural therapy according to Huneke (ref. 11), ampoules over nerve blocks according to Bracho (ref. 12) and segmental acupuncture according to Pistor (ref. 32). At the same time we want to give more credibility and respect to orthodox doctors as to therapists in natural medicine (ref. 1, ref. 8). In this way, we shall stimulate its use, so that the strategy of using these injections is developed in everyday practice.
Let us now go a little deeper:

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C. Biotherapeutics for local injections?

C.1. Subcutaneous injection

One of the most familiar ways of using biotherapeutics in everyday practice. Most of you use them in the oral form, as tablets or drops.
Although you may not be interested in giving injections in your practice, I think it is good to know more about the enormous possibilities of these biotherapeutic drugs. And it might give you a broader view on natural medicine, because injections can act in a more powerful way than oral medication.
The exact choice of the remedy itself (ref. 1, 2, 5).

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C.2. Acupuncture point injections

Those doctors familiar with acupuncture, can enhance their therapeutic effect by injecting intracutaneously or subcutaneously a biotherapeutic product instead of dry needling. This method was mentioned before by de La Fuye, Geyer, Matz, Frase and many other authors.
It is theoretically possible to inject a single remedy into an acupuncture point that is known for certain indications. This combination of homeopathy and Chinese medicine (homoeosinaty) was introduced by de La Fuye. I will give you some examples:

Chelidonium on Liver 13 for drainage of the liver,
Nux Vomica on Bladder 21 for gastritis,
Gnaphalium on Bladder 34 for ischias,
Sulfur on Bladder 52 for eczema,
Cantharis on Kidney 11 for cystitis,
Lobelia on Kidney 27 for asthmatic bronchitis,
Naja Tripudians on Bladder 17 for cardiac neurosis,
Crataegus on Heart 3 for palpitations,
Thuja on Gallbladder 30 for coxarthrosis,
Echinacea on Jenn Mo 19 for cough, asthma, bronchitis
Echinacea on Jenn Mo 22 (for cough, asthma, bronchitis),
Lachesis on Small Intestine 19 for vertigo,
Sulfur on Bladder 31 for climacteric disorders.

Cralonin on Heart 3 for palpitations,
Cralonin on Bladder 15 for palpitations,
Engystol on Small Intestine 14 for asthma,
Vertigoheel on Small Intestine 19 for vertigo,
Mullein pro injectione on Urinary Bladder 31 for climacteric disorders,
Traumeel on Gall bladder 20 for headache,
Spigelon on Gall bladder 20 for headache,
Zeel on Gall bladder 30 for pain in the hip region,
Pulsatilla composition on Jenn Mo 6 for activation of the defensive system.

It is also possible to inject a complex remedy into several points of an acupuncture meridian, like for example Discus compositum over the Governor vessel. Subcutaneous or intracutaneous injections are given on the mid-line of the back, or at the level of every vertebra in the region of the pain.
Those allopathic products should always be used in a diluted way (half a normal dose, adding several ml of physiological fluid and several ml of a local anesthetic), and should be injected with more care than the biotherapeutic one. I use them as a replacement for corticosteroid injections (see also ref. 51). Of course, some injections are given at the physician’s own responsibility, when the product is used in a way for which it has not been registered. Ticlodil is officially designed for the intravenous/intramuscular injection only and an oral Feldene is officially designed for the intramuscular injection only. So, I can not make any claims about their safety and efficacy, until more large-scale clinical studies are performed.

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F. Is it always necessary to give injections?

For those therapists who are not allowed to give injections, I can recommend to do trigger point therapy with stretch and spray, as Travell and Simons suggest. This is an easy and safe technique, and gives interesting results when dealing with several myofascial disorders. The patient is usually very enthusiastic, since he or she has almost immediate relief. Although this effect is temporary, long term effects are achieved with repeated sessions.
Even more interesting is ischemic compression. Ischemic compression is application of progressively stronger, painful pressure on a trigger point for the purpose of eliminating the trigger point’s tenderness and hyperirritability. Similar to acupressure and shiatsu, the thumb is used as the therapeutic tool. But we do not deal with acupuncture points, but solely with active trigger points, which can be found by clinical examination. The thumb action blanches the compressed tissues, which usually become flushed (hyperemic) on release of the pressure. The use of Traumeel ointment during and after the compression, enhances the effects of the treatment.
The clinical effects of the technique depend largely on the skills of the therapist. When looking for the active trigger points, wall plates can help. Textbooks (by Baldry, Travell and Simons) give more fundamental information on this subject. In Belgium, I am giving workshops on trigger point therapy, in order to show how this technique can be performed in everyday practice.

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Case:

Clinical examination shows a painful zone in the right brachioradialis muscle and a small spot at the epicondylus radialis, that is painful on digital pressure. He had ischemic compression on two trigger points in the brachioradialis muscle, twice a week.
He also got a local application of Arnica comp. ointment (e.g. Traumeel) in the right brachioradialis muscle and on that small painful spot at the epicondylus radialis, three times a day. Additional oral treatment with Ferrum-Homaccord, ten drops three times daily, gave complete relief after two weeks.

---

Case:

A girl of twelve has suffered a contusion of the lateral part of the thigh while snowboarding. The examination on the evening shows an obvious swelling of the thigh and an extensive haematoma. The region is very sensitive to pressure, and she refuses a local injection. I give her Arnica comp. ointment (Traumeel ointment, which was cooled in the fridge) and she applied it every hour the first day, every two hours the second and third day and four times a day the three next days. Putting the ointment in a refrigerator before application gives an extra cooling down in an acute situation. As an additional therapy, she received Arnica comp. tablets: I told her to take them at the same frequency as the application of the ointment. I asked her to keep the tablets in the mouth as long as possible, to give the product maximum resorption via the oral mucosa. After six days the pain and swelling were completely gone.

---

G. Conclusion

Although the use of corticoids is accepted as a standard technique in the treatment of several inflammatory diseases, we must recognize that specific injection techniques of biotherapeutic products are gaining more and more interest. Most physicians, however, are not familiar with these techniques. That is why I wanted to show the importance of these injections in the treatment of so-called inflammatory diseases.
By using biotherapics (drops or tablets), I have experienced that the combination of oral application (drops or tablets) and local treatment (ointment and/or injections) gives interesting results, both in acute as in chronic cases.

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by HEEL Medical – Scientific Department

What does neural therapy involve?

Neural therapy involves curative procedures which act via the autonomic nervous system. Techniques of neural therapy were developed from procedures applied for local anesthesis by the two brothers and general practitioners Dr. Ferdinand Huneke and Dr. Walter Huneke, both medical doctors in Düsseldorf.

Neural (Greek)

Of, relating to, or affecting a nerve or the nervous system.

Neuron

Nerve cell, nerve tracts, neuron theory

Neuron theory

The original version of the neuron theory, as published by Ramon y Cajal in 1934, stated that each nerve cell (i.e., neuron) represents an anatomical, genetic, functional, and regenerative unit.

The latest insights gained from electron-microscopic findings have confirmed the reality of synapses as physically and anatomically verified sites of junction: between two neurons (i.e., an interneuronal synapse) and between a neuron (peripheroreceptor) and the locus of action (i.e., a neuro-receptor synapse). These insights, in conjunction with complementary findings, represent the confirmed scientific basis for the entire fields of neurohistology, neurophysiology, and neuropathology.

The significance of neural therapy: Neural therapy involves curative techniques administered via the autonomic nervous system. The autonomic nervous system is a functional unit which includes the neurohumoral regulatory system in its overall mode of reflectoral operation. The autonomic nervous system encompasses the sympathetic nervous system, the parasympathetic nervous system, and the basic autonomic neural system. This basic autonomic neural system is the interstitial, soft connecting tissue which fills all organ interstices. It is composed of cells, nerves, capillaries, and extracellular fluid. This system plays the role of a communicator in the overall arrangement which enables each cell in the body to be in connection with every other cell. The autonomic nervous system is that part of the entire central nervous system which is of significance for the maintenance and reproduction of the particular organism involved. The autonomic nervous system primarily serves for control of the inner milieu. Since the control loops of the autonomic and somatic nervous systems are multiply interlinked at all synapse stages of the central nervous system, the functioning of the autonomic nervous system is not in fact strictly autonomous in nature.

The autonomic nervous system differs from the somatic nervous system in the following significant aspect:

• Connections to the periphery are not uniformly present.
• These connections are also subject to interruption by intermediate ganglionic stations (in which the ganglia are groups of nerve cell bodies). In the autonomic nervous system, sympathetic and parasympathetic ganglia are involved.

Human life is possible only in conjunction with the hermetic control of all regulation mechanisms. The pathways of the autonomic nervous system have the function of passing stimuli. Excessive stimuli disturb or block the development of energy and, in turn, the distribution of this energy. All neural therapeutic methods feature the introduction of energy to the impaired tissue, or they effect removal of the blockage by energy transfer. These procedures, however, also initiate reactions which are capable of eliminating damage which has already occurred. The body’s defense system, with its mechanisms of self-healing, is thereby stimulated to action.

All neural therapeutic techniques in the broad sense utilize this approach: acupuncture, chirotherapy, cutaneous and other types of stimulation, and cupping. And the same applies for neural therapy in the narrow sense: concerted injection therapy with local anesthetic.

HISTORICAL SUMMARY OF NEURAL THERAPY

Neural therapy by means of local anesthetic was discovered orientationally by Ferdinand and Walter Huneke. The Huneke brothers, both medical doctors from a family of physicians, had attempted unsuccessfully for years to treat their sister’s migraine attacks.

Atrophanyl, a new medication for rheumatism, was introduced onto the market in 1925. On the advice of a colleague, Ferdinand Huneke treated his sister by intravenous injection of the new preparation. The therapy immediately interrupted the migraine attack, with all its accompanying symptoms. Only one single subsequent administration, with the same success, was required to completely free his sister of her long-term suffering.

The two brothers were naturally impressed by the success of their treatment, but they could not explain the effects. They eventually determined, however, that Atrophanyl was available in the following two types:

• For intravenous administration, without 2% addition of cocaine
• For intramuscular administration, with 2% addition of cocaine

In his treatment of his sister, Ferdinand had confused the two solutions and had mistakenly administered the procaine solution intravenously – a form of administration which had until that time been expressly forbidden. It was believed then that procaine could damage important centers in the brain.

Motivated by the unexplained success of the therapy, the brothers then began to separately study the causative factors behind the accidental cure.

The two brothers independently discovered that the positive effect of the procaine was not only associated with the mode of administration (i.e., intravenous injection), but that the determining criterion for the therapeutic results was most probably the specific point of the injection. They wondered whether previously unknown reflex-type reactions could possibly act via Head’s zones.

Beginning with these findings, Ferdinand and Walter Huneke used injections administered at particular points to treat conditions of pain in the respectively segment-associated areas of the body. They named their technique “therapeutic anesthesia,” until Kibler, a friend, proposed a better name: “segment therapy with local anesthesia.”

In 1928, the Huneke brothers published their findings in Medizinische Welt under the title, “Unexpected Remote Therapeutic Effects of Local Anesthesia.”

Neural therapy as developed by the Hunekes is administered in two forms:

• A. Segment therapy: Concerted injections of anesthetic into the segmental area associated with the illness.
• B. Elimination of a focal disorder: Rendering a causal focal disorder ineffective by means of a neural therapeutic agent acts as causal therapy, with the elimination of complaints and pain in seconds (the so-called Huneke phenomenon), including pain without segmental association.

Definition of focal disorder

A focal disorder is chronically altered tissue which causes remote disorders via neural paths. A focal disorder is considered to be any pathological alteration which possesses the ability to cause remote disorders beyond its immediate locale.

Location of focal disorders

Pinpointing of the focus responsible for a disorder begins with preparation of a concerted case history, and includes systematic injections at potential foci.

SEGMENT THERAPY

Segment therapy is based on the insight that all parts of a bodily segment respond by reflection, as a unit, to particular processes. Stimulation pulses proceed from the body’s periphery, via the spinal cord, to the segment-associated organ, and vice versa. Pulses also travel along the dermato-visceral reflex path, or from an organ via the spinal cord to other organs—as well as along the viscero-visceral reflex path.

IMPORTANT: In all cases it is the entire human being which becomes ill, and never only one isolated organ. Concerted neural therapy not only interrupts pathological reflex paths, but also normalizes all autonomic neural functions through repolarization of the stimulus-disordered cell-membrane potentials.

• Most important therefore is the point at which the injection is performed, and only to a lesser degree the particular medication which is administered. Segment therapy is genuine treatment in the original sense of the Latin origin “tractare,” to handle—a laying on of the hand.

Head’s zones

These zones were named for the work of Sir Henry Head, a London neurologist (1861–1940). These are hyperesthetic-hyperalgesic zones which appear on the surface of the human trunk in conjunction with disorders of certain inner organs, and which are characterized by heightened cutaneous sensitivity. This phenomenon has been explained by the fact that these cutaneous zones are provided with their sensitive innervation (nerve supply) from the same neural segments which supply the disordered organ.

Disordered sensitivity is also often found together with alterations of muscle tone (defense musculaire—for example, appendicitis), involving the viscero-cutaneous reflex.

What is meant by “chronically altered tissue”? Non-vital teeth, points of focal infection, tonsils as foci, scars on the surface of the skin or deeper in the body, scars on bones, foreign bodies, chronically inflamed organs (e.g., appendicitis chronica), and residual conditions after inflammatory processes which have subsided; e.g., in the ears and sinus cavities, at the gall bladder, at the appendix, in the female genital tract, and in the prostate.

HUNEKE’S INSTANTANEOUS PHENOMENON

In 1941, Ferdinand Huneke discovered that there are foci located on neural paths and not subject to any segmental assignment, yet capable of initiating and maintaining certain illnesses. These foci can be rendered ineffective in the neural-therapeutic sense by the concerted administration of local anesthetics. The disorders caused by the treated foci will then immediately vanish as a result of Huneke’s so-called flash phenomenon.

Huneke’s flash phenomenon is involved if all three of the following conditions are met:

1. All symptoms vanish instantaneously and completely.
2. Freedom from the symptoms continues for at least twenty hours.
3. The above phenomena are repeated with recurring symptoms.

If all conditions are met, injections are continued at intervals of one week—with the periods between injections gradually lengthened—until a complete cure is achieved.

Important: If, however, significant improvement is not achieved by injections into the segment, and if the flash phenomenon is not observed in the suspected focus, then injections at this point are futile, and the search for the proper focus must continue.

N.B.: The most important factor in neural therapy is the point of injection, and not the particular medication injected.

THE TECHNIQUES OF NEURAL THERAPY

Instruments required

• Single-use cannulae
• Single-use syringes (5‑ml plastic syringes have proved most effective for focal disorders)
• A neural therapeutic agent.

Dosage and manner of application will vary considerably from case to case.

Wheal test

Raised, beetlike efflorescence with a red areola, as the expression of an acute edema of the skin.

Wheal test method: If a wheal applied intracutaneously with 0.2 ml of isotonic saline solution is no longer evident after 40 to 45 minutes, the patient has a propensity to develop edema. Disappearance of the wheals within 3 to 30 minutes indicates pronounced edema tendency.

DEFINITIONS

• Neural therapy: Treatment of a disease via the autonomic nervous system.
• Autonomic nervous system: A functional unity which includes the entire conditional-reflectorally functioning neuro-humoral system of regulation.
• The autonomic nervous system includes:
  • The sympathetic nervous system
  • The parasympathetic nervous system
• Basic autonomic neural system: An interstitial (i.e., located in intermediate tissue), soft connecting tissue which fills the interstices between organs. It is composed of cells, nerves, capillaries, and the extracellular liquid space. This system performs communicative functions required to provide connection between each cell of the body and every other cell.
• Segment therapy: Therapy administered in a segment via Head’s zones. Deeper-lying organs are accessed via dermato-visceral reflex paths (as defined by Pischinger).
• Focal therapy: If no success is achieved after repeated local or segment therapy, it may be assumed that a focal disorder is responsible for the complaint. It is then necessary to search for the responsible focus.
• Local therapy: Direct administration of neural therapeutic agents which enables long-term freedom or relief from symptoms.
• Procaine: p-aminobenzoyl diethylaminoethanol hydrochloride; the most frequently used local anesthetic; weak in its effects, non-toxic, and not harmful to human tissues.
• Local anesthetic: An agent which, when locally administered, reversibly eliminates the excitability and conductivity of nerves or their endings. The area involved then becomes insensitive to pain.
• Local anesthesia: Insensitiveness to pain in a locally restricted area of the body.

Activates the lymphatic system

The efficacy of Lymphomyosot for the treatment and prevention of lymphatic disorders has been demonstrated in numerous studies¹–⁵.

Extremely well tolerated

In a study of 3,512 patients, including 1,124 (32%) children under 10 years of age, 99.8% assessed Lymphomyosot therapy as “very well tolerated” throughout the course of treatment¹.

3 different dosage forms

• Drops: 15 drops 3 times per day
• Tablets: 3 tablets 3 times per day
• Ampoules: 1 ampoule 1 to 3 times per week

As a standard for comparison, 10 infusions with 600 mg of α-lipoic acid over 8 months improved tactile sensitivity among study patients by only Ø +0.9. However, a combination therapy with Lymphomyosot (15 drops twice daily for 8 months) and α-lipoic acid (10 infusions with 600 mg of α-lipoic acid over 8 months) improved tactile sensitivity by Ø +3.25⁵.

• Neural nutrient insufficiency
• Diminished tactile sensitivity
• Defence reaction absent
• Risk, imbalance, damage
• Amputation
• Gangrene
• Diabetes
• Diabetic foot
• Inflammation
• Malfunction/glycosylation of the matrix
• Peri-neural oedema

Reduction of oedema

Severity of symptoms	Start	Reduction	No reduction
Slight	14	10	4
Moderate	26	19	7
Severe	0	9	1

50 patients diagnosed with diabetic neuropathy were administered 15 drops of Lymphomyosot twice daily for 8 months. At the end of the treatment period, lymphoedema and tactile sensitivity had profoundly improved⁵.

Improvement of tactile sensitivity

Severity of symptoms	Start	End	Difference
Slight	5.0	7.5	+2.5
Moderate	3.0	6.0	+3.0
Severe	0.0	3.0	+3.0

Minimizes the risks associated with diabetic neuropathy

• Improved matrix metabolism
• Improved tactile sensitivity
• Eliminated pain in 75% of patients
• Lymphatic disorders
• Recurrent infections
• Detoxification and drainage

Lymphomyosot – recommended for

• Lymphhoedema (i.e. for promoting postmammectomy drainage)
• A compromised defence system (i.e. for treating recurrent and acute infections, and inflammatory disorders such as tonsillitis or lymphangitis)
• Tonsillar hypertrophy
• Lymphadenitis
• Systemic toxicities (i.e. for treating chronic diseases or amalgam-related disorders)

Composition:

• Drops: 100 g cont.: Myosotis arvensis D3, Veronica officinalis D3, Teucrium scorodonia D3, Pinus silvestris D 4, Gentiana lutea D 5, Equisetum hyemale D4, Sarsaparilla D6, Scrophularia nodosa D3, Juglans regia D3, Calcium phosphoricum D12, Natrium sulfuricum D4, Fumaria officinalis D4, Levothyroxinum D12, Araneus diadematus D6 5 g each; Geranium robertianum D4, Nasturtium officinale D4, Ferrum jodatum D12 10 g each. Contains 35 vol.-% alcohol.
• Tablets: 1 tablet cont.: Myosotisarvensis D3, Veronica officinalis D3, Teucrium scorodonia D3, Pinus silvestris D4, Gentiana lutea D5, Equisetum hyemale ex herba rec. D4, Sarsaparilla D6, Scrophularia nodosa D3, Juglans regia D3, Calcium phosphoricum D12, Natrium sulfuricum D4, Fumaria officinalis D4, Levothyroxinum D12, Araneus diadematus D6 5 g each; Geranium robertianum D4, Nasturtium officinale D4, Ferrum jodatum D12 10 g each.
• Injection solution: 1.1 ml cont.: Myosotis arvensis D3, Veronica officinalis D3, Teucrium scorodonia D3, Pinus silvestris D4, Gentiana lutea D5, Equisetum hyemale D4, Sarsaparilla D6, Scrophularia nodosa D3, Juglans regia D3, Calcium phosphoricum D12, Natrium sulfuricum D4, Fumaria officinalis D4, Levothyroxinum D12, Araneus diadematus D6 0.55 µl each; Geranium robertianum D4, Nasturtium officinale D4, Ferrum jodatum D12 1.1 µl each.

Indications: Status lymphaticus (tendency to hyperdevelopment of the lymphatic organs; tendency to development of oedemas and infections); glandular swelling; tonsillar hypertrophy; chronic tonsillitis.
Contraindications: In case of thyroid disorders, this preparation may not be administered without prior approval from a physician.
Side effects: None known.
Interactions with other medication: None known.
Dosage: Drops: In general, 15-20 drops 3 times daily. Tablets: In general, 3 tablets to be dissolved in the mouth 3 times daily. Injection solution: In acute disorders daily, otherwise 3-1 times weekly 1 ampoule i.m., s.c., i.d.
Package sizes: Drops: Drop bottles containing 30 and 100 ml. Tablets: Packs containing 50 and 250 tablets. Injection solution: Packs containing 10, 50 and 100 ampoules of 1.1 ml.
Revised: April 2001.

References

1. Zenner S, Metelmann H: Therapeutic Use of Lymphomyosot® – Results of a Multicentre Use Observation Study on 3,512 Patients. Biological Therapy 1990, Vol Vlll No. 3.: 49–53,67–72, and Vol Vlll, No. 4: 79–84, 94
2. Küstermann K, Weiser M: Treatment of a Lymphatic Disease with Homoeopathic Therapy. Biologische Medizin 1997; Vol 26 No. 3: 110–114
3. Rinneberg A-L: Lymphomyosot®: The Therapy of Tonsillitis and Prophylaxis against its Recurrence. Biological Therapy 1991; Vol IX No. 1: 111–114
4. Riley D, White S: A Biotherapeutic Approach to the Treatment of Inflammatory Disorders: A Drug Monitoring Trial. Biological Therapy 1992; Vol. X No. 3: 267–271
5. Dietz R: Possibilities of Lymph Therapy with Diabetic Polyneuropathy. Biologische Medizin 2000; Vol 29 No. 1: 4–9

Rolf Gebhardt*

Institute of Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany

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ARTICLE INFO

Article history:

• Received 21 May 2008
• Received in revised form 11 December 2008
• Accepted 18 January 2009
• Available online 31 January 2009

Keywords:

• Antioxidants
• Apoptosis
• Cadmium
• Cytochrome C
• Hepatoprotection
• Plant tinctures

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ABSTRACT

Cadmium is a heavy metal of considerable environmental concern that causes liver damage. This study examined the possible prevention of cadmium toxicity in human HepG2 cells and primary rat hepatocytes by Hepeel®, a combination preparation of tinctures from seven different plants. Hepeel® prevented cadmium chloride (CdCl₂)-induced cell death in both HepG2 cells and hepatocytes, and also reduced the loss of glutathione, lipid peroxidation, nuclear fragmentation, caspase activation and release of mitochondrial cytochrome C. To compare their relative efficacy, the seven constituent plant tinctures of Hepeel® were also separately tested. The tinctures China and Nux moschata, which exert solely anti‑oxidative effects, failed to reduce cytotoxicity, and only protected against loss of glutathione and lipid peroxidation. In contrast, the tinctures Carduus marianus and Chelidonium, demonstrated anti‑apoptotic effects, and protected HepG2 cells and primary hepatocytes against CdCl₂-induced cell death. These results demonstrate how the effectiveness of Hepeel® is determined by the synergistic features of its constituent tinctures. Furthermore, we conclude that cadmium toxicity in the liver is mainly due to stimulation of the intrinsic apoptotic pathway, but may be intensified by increased oxidative stress.

© 2009 Elsevier B.V. All rights reserved.

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1. Introduction

Environmental exposure to fluctuating concentrations of heavy metals poses an enormous challenge for biological organisms. Toxic metals cause a vast array of adverse effects, including neurotoxicity, hepatotoxicity and carcinogenicity (Waalkes et al., 2000; Godt et al., 2006). Due to the global dispersion of heavy metals and their extensive use in modern society, some human exposure to toxic metals is inevitable. This ongoing prevalence of metal exposure necessitates protective measures at the environmental, social and individual level.

Cadmium is one of the most common toxic heavy metals, due to its primary accumulation in the liver and kidney (Godt et al., 2006). Cadmium causes hepatic, renal, skeletal, respiratory, and vascular disorders in humans (Nordberg, 1992; Waalkes et al., 2000), and it may also affect Leydig cells of the testes and hepatocytes and stellate cells of the liver (Koizumi et al., 1992; Dudley and Klaassen, 1984; Fariss, 1991; Souza et al., 2004a,b). Furthermore, cadmium is a potent carcinogen (Godt et al., 2006).

There is growing evidence that the oxidative stress (Sarkar et al., 1995) via reactive oxygen species (ROS) generation and mitochondrial damage are among the basic mechanisms of cadmium toxicity (Sarkar et al., 1995).

The combination preparation Hepeel® is frequently used to stimulate liver function and improve antioxidant function in acute and chronic diseases, such as cholangitis and cholecystitis (Gebhardt, 2003). Hepeel® also demonstrates several other protective features, such as induction of glutathione-S-transferase activity (Gebhardt, 2003). These findings prompted the present investigation of the hepatoprotective potential of Hepeel®, and its seven constituent plant tinctures, against cadmium-induced hepatocellular damage. To thoroughly examine this, and to provide comparative experimental data for two different cell types, we used the human hepatoblastoma cell line HepG2 and primary rat hepatocytes. Exposure to Hepeel® largely prevented cell death, and oxidative and apoptotic pathomechanisms were differentially affected by the constituent tinctures. The combined anti-oxidative and anti-apoptotic properties of Hepeel® and its constituent tinctures support its overall protective effect against cadmium-induced toxicity in liver cells.

2. Materials and methods

2.1 Materials

Hepeel® tinctures were prepared from seven different plants, according to procedures 3 and 4 of the German Homeopathic Pharmacopoeia (HAB, 2000), and were provided by the Biologische Heilmittel Heel GmbH (Baden-Baden, Germany). The following seven constituent tinctures were used: (1) Chelidonium majus, prepared from Chelidonium majus L. (Ch-B 007009, 10⁻² dilution), (2) Carduus marianus, prepared from Silybum marianum L. (Ch-B 007034, 10⁻² dilution), (3) Verratrum album L. (Ch-B 007050, 10⁻³ dilution), (4) Colocynthis, prepared from Citrullus colocynthis L. (Ch-B 007058, 10⁻³ dilution), (5) Lycopodium, prepared from Lycopodium clavatum L. (Ch-B 007001, 10⁻³ dilution), (6) Nux moschata, prepared from Myristica fragrans Houtt (Ch-B 007026, 10⁻³ dilution), and (7) China, prepared from Cinchona pubescens, Vahl (Ch-B 007018, 10⁻³ dilution). Hepeel® is a combination of all tinctures at the dilutions given above, with the addition of Phosphorus, a 10⁻³ dilution of yellow phosphorus. Hepeel® was supplied in sterile ampoules by Biologische Heilmittel Heel GmbH. The relative volume composition of 1:1 mL Hepeel® injection solution is: Chelidonium majus, 10⁻³ dilution) 1.1 μL; Carduus marianus (Silybum marianum, 10⁻³ dilution) 0.55 μL; Verratrum album, 10⁻² dilution) 2.2 μL; Colocynthis (Citrullus colocynthis, 10⁻³ dilution) 3.3 μL; Lycopodium clavatum, 10⁻² dilution) 1.1 μL; Nux moschata (Myristica fragrans, 10⁻³ dilution) 1.1 μL; China (Cinchona pubescens, 10⁻³ dilution) 1.1 μL and Phosphorus (white phosphorus 0515.41).

Dichlorodiphenyltrichloroethane (DDT) was purchased from Sigma (Daisenhofen, Germany). All other chemicals were from Roche Diagnostics (Mannheim, Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany) or Sigma (Daisenhofen, Germany). Cell culture plates with tissue culture filter inserts were from Techno Plastic Products AG (Trasadingen, Switzerland).

2.2 Culture of HepG2 cells

HepG2 hepatoblastoma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Eggenstein, Germany) supplemented with 2 mM glutamine, 10% fetal calf serum, 40 U/mL streptomycin and 50 U/mL penicillin, as previously described (Gebhardt, 1997). Cells were passed weekly, when confluent. Cell stocks (passage ≤31 till 40) were kept frozen in liquid nitrogen. Frozen cells were thawed, cultured for one week, and passed at least once before use. Confluent HepG2 cell cultures were used for all experiments.

2.3 Preparation and culture of rat hepatocytes

Sprague-Dawley rats were bred and maintained at the Medizinisch-Experimentelles Zentrum at the University of Leipzig, according to local ethical rules for animal care. They were kept on normal maintenance diet V1534 (Sniff, Soest, Germany) and tap water, ad libitum. Primary hepatocyte cultures were prepared by the livers of male rats (200–310 g) with collagenase perfusion, as previously described (Gebhardt, 1997). Cells were cultivated in Williams medium E (Lonza, Verviers, Belgium) on collagen-coated plastic plates, at a uniform cell density of 125,000 cells/cm². During the first 2 h, culture medium was supplemented with 10% fetal calf serum, and culture medium was used thereafter. The medium volume was maintained at 100 μL/cm² of plating area. Additional details of cell culture have been reported elsewhere (Gebhardt, 1997; Gebhardt et al., 1994). For toxicity experiments, incubation in various agents usually started 2 h after plating.

2.4 Induced toxicity with cadmium chloride

The nominal concentration of CdCl₂-induced cytotoxic effects was different for each cell type. For HepG2 cells, culture medium was supplemented with concentrations ranging from 3 to 8 μM. For primary rat hepatocytes, concentrations ranged from 2 to 6 μM. The highest CdCl₂ concentrations caused the greatest cell death in each cell type. In HepG2 cells, 8 μM CdCl₂ caused about 52% cell death, within 30 h of incubation, in hepatocytes, 6 μM CdCl₂ caused 72% cell death within 24 h of cultivation.

2.5 Preparation of Hepeel® tinctures

To prepare a working dilution of each tested compound, one part Hepeel® or tincture was mixed with 5 parts v/v of serum-free Williams Medium E, and gently shaken for 20 min at room temperature. This working solution of effective 0.1 dilution was used for further dilutions with Williams Medium E as specified in figure legends. Appropriate controls replaced each tincture or Hepeel® with equal volumes of ethanol.

2.6 Determination of cytotoxicity

Cytotoxicity of the tested compounds was determined using the colorimetric MTT-assay (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), as previously described (Gebhardt, 1997).

2.7 Determination of lipid peroxidation and ROS production

Malondialdehyde (MDA) measurements were used to quantify lipid peroxidation (Gebhardt, 1997). HepG2 cells or rat hepatocytes seeded on 60 mm petri dishes were incubated with or without CdCl₂ (3 or 4 μM) for 60 min after 30 h and 24 h of cultivation, respectively. In order to enhance oxidative stress, some plates were simultaneously exposed to t‑butyl hydroperoxide (t‑BHP; final concentration 1.5 mM). Thereafter, cells were washed with 0.9% NaCl, resuspended, and scraped into 1 mL of 50 mM potassium phosphate buffer (pH 7.4), then homogenised by sonication for 10 s (15 μs maximal sonpower, Sonipuls HD 2200, Bandelin electronic, Berlin, Germany). MDA was determined by thiobarbituric acid (TBA) assay (Esterbauer and Cheeseman, 1990; Gebhardt, 1997). The protein content of homogenates was measured following the procedure of Lowry et al. (1951).

Measurement of intracellular ROS was accomplished by using the DCFH assay (Wang and Joseph, 1999). HepG2 cells or rat hepatocytes cultivated on collagen-coated 95-well black flat bottom plates were washed 3 times with Krebs‑Ringer‑HEPES (KRH) solution pH 7.2 (Pavlica and Gebhardt, 2005). Cells were preloaded with 0.1 mM DCFH in either DMEM (HepG2 cells) or Williams Medium E (rat hepatocytes) for 30 min, then washed 3 times with KRH buffer. Cells were then treated simultaneously with CdCl₂ (3 μM) and the test compound diluted 1:10 with different starting dilutions (indicated in Table 1) for an additional 30 min. Fluorescence (485/520 nm, excitation 485/emission 520 nm, microplate reader, TECAN) was recorded for up to 30 min, while temperature was maintained at 37 °C. Percentage increase in fluorescence units/well was calculated by the formula: F₃₀/F₀ × 100, where F₃₀ = fluorescence at time 30 min, and F₀ = fluorescence at time 0 min (Pavlica and Gebhardt, 2005).

2.8 Determination of cellular glutathione content

To measure cellular glutathione (GSH) content, cells were cultured in 6-well plates for 30 h (HepG2 cells) or 24 h (primary rat hepatocytes). Test compounds were added 2 h after plating, along with the first change of medium. At the end of the incubation period, cells were washed and scraped into HEPES buffered isotonic medium as previously described (Pavlica and Gebhardt, 2005). Determination of GSH content was performed according to the method of Gebhardt and Faustel (1997).

2.9 Detection of apoptotic nuclei with DAPI

The blue nuclear dye DAPI (4′,6-Diamidino-2-phenylindole) was dissolved in methanol at 5 μg/mL and stored as stock solution. Cells were washed twice in potassium phosphate buffer (PBS) and fixed with ice-cold methanol. Thereafter, a working solution of DAPI (1 μg/mL) in methanol was added, and cell nuclei were stained for 15 min at 37 °C. Destaining was achieved by replacing methanol with pure methanol, followed by two rounds of washing with PBS.

2.10 Determination of caspase activity

Measurement of caspase-3 activity was based on the cleavage of a colorimetric substrate determined by the increase in absorbance at 405 nm. The assay was performed according to the instructions of the manufacturer (caspase-3 activity assay kit; Oncogene, Bad Soden, Germany) and adapted for HepG2 cells as described by Ochiai et al. (2004). Recombinant caspase-3 was used for assay calibration.

2.11 Preparation of cellular fractions and Western blot analysis

To measure cytochrome C release, cellular extracts were prepared by lysing cells in 10 mM Tris-buffer (pH 7.4) containing 2 mM EDTA, 1 μM pepstatin, 1 mM PMSF, leupeptin, 100 μM PMSF (phenylmethylsulfonyl fluoride), and 250 mM sucrose. Cells were homogenized by repeated passage through a 26-gauge needle, and were centrifuged at 14,000 × g for 10 min at 4 °C. Cytosolic supernatants and pellets containing mitochondria were collected and analyzed for spectral concentrations of mitochondrial protein, then used for Western blot analysis as previously described (Haupt et al., 2000). Cytochrome C was detected using a cytochrome C (Ab-8) antibody (sc-13156, Santa Cruz Biotechnology Inc., Heidelberg, Germany) followed by alkaline phosphatase-conjugated secondary antibody.

2.12 Statistical evaluation

Data were analysed for significance with a Student’s t-test for comparisons between two groups. Data are presented as mean ± standard deviation (SD) of three to four measures, except when stated otherwise.

3. Results

3.1 Cytotoxicity of cadmium chloride on hepatocellular populations

The cytotoxic effect of CdCl₂ on HepG2 cells was concentration- and time-dependent. Within the first 24 h of exposure, HepG2 cells tolerated up to 5 μM CdCl₂, but quickly died at higher concentrations (Fig. 1). At 7 μM CdCl₂, almost all cells were dead or had detached from the substrate. At 5 μM CdCl₂ or below, no visible alterations in cell morphology and nuclei were detectable after 24 h (data not shown). However, deterioration was seen at 5 μM CdCl₂ when cultivation was continued for another 6 h (Fig. 1). At that time, cadmium-induced cytotoxicity was already apparent at lower concentrations. The first signs of cytotoxic influence were detected above 2 μM, and almost all cells died at a concentration of 5 μM, as determined by MTT reduction to less than 10% of controls. The EC₅₀-value for CdCl₂-induced cytotoxicity in HepG2 cells was determined to be 5.9 μM after 24 h, and 2.8 μM after 30 h of cultivation.

Rat hepatocytes were even more sensitive to cadmium, and cytotoxicity was more prominent than in HepG2 cells, at all culture times. At 24 h after addition of CdCl₂, MTT reduction was already decreased in a concentration-dependent manner, above 2 μM doses (Fig. 2). At 6 μM, absorbance was reduced by approximately 70%. The EC₅₀-value for CdCl₂-induced toxicity was 3.7 μM. After 30 h, cell detachment in the MTT assay had further dropped, and were lower than those of HepG2 cells at all concentrations of cadmium (data not shown). Therefore, all subsequent measurements of cell viability were performed in HepG2 cells at 30 h of cultivation, and in rat hepatocytes at 24 h of cultivation.

3.2 Protection against cadmium cytotoxicity by Hepeel® and constituent tinctures

In the presence of Hepeel®, cadmium cytotoxicity was reduced in both cell types. In HepG2 cells at 30 h of culture, Hepeel® application resulted in the gradual increase of viability from 32% (control) to 53%, as dilutions changed from 10⁻³ to 10⁻¹ (Fig. 3A). At the 10⁻² dilution, there was significant enhancement of viability (P < 0.01).

Among the constituent tinctures, only Carduus marianus and Chelidonium, were effective in reducing cadmium cytotoxicity (Table 1). Within the range of 10⁻⁵ to 10⁻³ dilutions, Carduus marianus caused increased cell viability in a concentration-dependent manner, to values between 60 and 70% (Fig. 3B). Chelidonium application resulted in maximal values slightly above 60% (Fig. 3C). Also, the cell sensitivity was slightly higher with Carduus marianus, and significant differences were seen starting at the 2.5 × 10⁻⁴ dilution, whereas with Chelidonium significant differences were not apparent until the more concentrated dilutions of 10⁻⁴ and lower.

Similar results were obtained with rat hepatocytes after 24 h of cultivation. The 10⁻³ dilution of Hepeel® increased viability from 68% to almost 88%. At the same 10⁻³ dilution, Carduus marianus reached 95% and Chelidonium increased 87% viability (Table 1). As for HepG2 cells, the other constituents of Hepeel® did not reduce cytotoxicity (Table 1).

3.3 Cadmium-induced lipid peroxidation and ROS production

Exposure of HepG2 cells to CdCl₂ for 24 h did not change the rate of lipid peroxidation, as evidenced by the unchanged cellular production of malondialdehyde (MDA) compared to control measures (Table 2). However, when challenged with 1.5 mM t‑BHP, HepG2 cells exposed to 3 μM CdCl₂ responded with a 2.1-fold increase, and those exposed to 4 μM responded with a 2.3-fold increase of MDA, compared to control cells not exposed to cadmium.

Likewise, ROS production of HepG2 cells detected by DCFH fluorescence was stimulated by CdCl₂ only in the presence of t‑BHP (Table 2). The relative increase in ROS production was comparable to that for lipid peroxidation.

As shown in Table 3, Hepeel® significantly reduced t‑BHP-induced MDA production in both untreated HepG2 (control) cells and HepG2 cells exposed to CdCl₂ for 24 h. Among all tinctures, Carduus marianus was the most effective (Table 3). Dilutions were almost as broad.

Likewise, ROS production of HepG2 cells was reduced in a pattern similar to that of MDA measures: Hepeel® (31%), Carduus marianus (36%), China (18%), and Nux moschata (16%). All other tinctures were ineffective at reducing the CdCl₂-induced MDA production (data not shown).

The results for rat hepatocytes were different. In these cells, CdCl₂ led to an increase of MDA production of 55%, and an increase in ROS production of 32%, compared to control hepatocytes exposed to cadmium. However, as in HepG2 cells, the sensitivity to t-BHP in the presence of cadmium was also increased approximately 2-fold, from 155% to 302% for MDA, and from 132% to 273% for ROS. The following agents significantly counteracted the impact of CdCl₂, as apparent via the following reduction in MDA measures: Hepeel® (31%), Carduus marianus (36%), China (18%), and Nux moschata (16%). All other tinctures were ineffective at reducing the CdCl₂-induced MDA production (data not shown).

3.4 Cadmium-induced loss of GSH

A moderate drop in cellular GSH (19 ± 5%) was observed in HepG2 cells in response to exposure to CdCl₂ at a concentration of 3 μM (Table 4). This value is in accordance with an EC₅₀-value of approximately 4.5 μM. This loss was considerably enhanced (55 ± 4%) when cells were additionally exposed to t‑BHP. Only Hepeel® and the tinctures Carduus marianus, China and Nux moschata were able to significantly counteract the influence of CdCl₂, with or without additional t‑BHP (Table 4). When used alone, Hepeel® and Carduus marianus were able to completely restore cellular GSH content.

3.5 Cadmium-induced apoptosis

Cadmium toxicity via apoptosis was measured by DAPI-staining in two different ways; counting of fragmented nuclei and monitoring of cell death. Within 30 h of 3 or 4 μM CdCl₂ exposure, apoptotic fragmentation in HepG2 cell nuclei was apparent after DAPI staining, and total cell numbers were decreased (Fig. 4). Specifically, the percentage of apoptotic nuclei increased from less than 0.1% (controls) to about 8% in the presence of 3 μM CdCl₂ (Table 5). At earlier time points, such as 24 h, the proportion of fragmented nuclei was lower than at 30 h.

Addition of Hepeel® to the culture medium considerably reduced the apoptotic response at all concentrations of CdCl₂ in HepG2 cells and hepatocytes (Table 5). This influence was particularly pronounced in hepatocytes exposed to 4 μM CdCl₂, wherein the proportion of apoptotic nuclei was diminished from 42% to 4% (Table 5). A similar but less pronounced effect of Hepeel® could be observed in the presence of 5 μM CdCl₂ (cf. Fig. 5D).

Similar to the results seen in the MTT assays, the co-application of either Carduus marianus or Chelidonium with CdCl₂ effectively reduced the number of apoptotic nuclei, and enhanced cell survival (Fig. 4C and D). In the presence of 4 μM CdCl₂ and 10⁻⁴ final tincture dilutions, the proportion of fragmented nuclei in hepatocytes was 7% for Carduus marianus and 11% for Chelidonium (Table 5).

3.6 Cadmium-induced activation of caspases

Results for caspase-3 activity measurements were similar to those for apoptosis. In HepG2 cells, 3 μM CdCl₂ induced a significant increase in caspase-3 activity within 24 h (Table 6), with a 1.8-fold increase in caspase-3 and a 2.5-fold increase of caspase activity as measured by caspase-3/7 assay. Simultaneous addition of Carduus marianus at a 10⁻⁴ dilution to the culture medium resulted in a decrease of caspase-3 activity to about 1.3-fold, and the 10⁻³ dilution decreased caspase-3 activity to about 1.2-fold, relative to the vehicle-treated controls. Chelidonium was slightly less effective, but still reduced caspase-3 activity significantly in both assays (Table 6). A similar result was obtained for the Hepeel® 10⁻⁴ dilution, which reduced CdCl₂-induced caspase activity in both assays by approximately 40% (Table 6). Aside from Carduus marianus and Chelidonium, none of the other constituent tinctures was effective (not shown), since many HepG2 cells detached or decomposed completely within 30 h of CdCl₂ exposure.

3.7 Cadmium-induced release of cytochrome C

The release of cytochrome C from mitochondria of HepG2 cells was significantly higher in the presence of 3 μM CdCl₂ than in unexposed cells, which showed almost no release (Fig. 6). Densitometric analysis revealed a 27-fold increase in cytochrome C in CdCl₂-treated versus vehicle control cells. Hepeel® (10⁻¹) reduced the release of cytochrome C by about 5-fold, and Carduus marianus (10⁻³) reduced it by 7-fold (Fig. 6). Chelidonium was almost as effective as Carduus marianus, while treatment with China showed no effect (data not shown).

4. Discussion

Our results demonstrate a strong protective effect of the combination preparation Hepeel® and several of its constituent plant tinctures against cadmium-induced hepatocellular damage in both human hepatoblastoma cell line HepG2 and primary rat hepatocytes. We showed that cadmium-induced hepatocellular damage is effectively counteracted by these agents, thus gaining insight into potential mechanisms of this protective effect, which focus on two aspects: oxidative stress, and occurrence of apoptosis.

There are conflicting reports in the literature about oxidative stress during cadmium cytotoxicity. While some authors report that cadmium toxicity is due to, or at least associated with, increased oxidative stress and lipid peroxidation (Dudley and Klaassen, 1984; Fariss, 1991; RIkans and Yamano, 2000; Souza et al., 2004a,b; Koizumi et al., 2006), other authors could not detect enhanced lipid peroxidation in response to cadmium exposure in vivo and in vitro (Harvey and Klaassen, 1983; Aydin et al., 2003).

Concerning the occurrence of apoptosis in response to cadmium exposure our results are consistent with findings in mouse and rat liver (Habeebu et al., 1998; Pourahmad et al., 2001; Li and Lim, 2007) as well as human hepatocytes (Lasfer et al., 2008), and corroborates similar conclusions based on the observation of DNA laddering and other markers of apoptosis in response to cadmium exposure in HepG2 cells (Aydin et al., 2003; Oh and Lim, 2006).

Our results with DAPI staining also showed that treatment with Hepeel® and the single plant tinctures, which protected against cadmium toxicity, reduced the number of apoptotic nuclei. Furthermore, these agents also inhibited the activation of pre-apoptotic caspases and the release of mitochondrial cytochrome C. Therefore, these results strongly suggest that the most effective single tinctures, Carduus marianus and Chelidonium, are able to counteract intracellular processes other than oxidative stress, such as events leading to caspase activation and subsequent apoptosis, in response to cadmium. Silybin is an active substance in the Carduus marianus tincture, and is known to exert an anti-apoptotic influence in other systems (Singh and Agarwal, 2004; Pock et al., 2006). Thus, silybin may contribute to the protective effects of the tincture. However, direct anti-apoptotic properties of Chelidonium have not yet been described. Interestingly, alkaloids derived from Chelidonium such as chelerythrine and sanguinarine interact with the cytoskeleton (Slaninova et al., 2001), and of these, the alkaloid chelerythrine is an inhibitor of protein kinase C (Herbert et al., 1999). In addition, chelerythrine recently described as an inhibitor of BclXL function, which may help explain the pro-apoptotic effect observed with Chelidonium (Chan et al., 2003). In fact, detailed studies on the molecular interactions of chelerythrine revealed binding sites distinct for the Bcl3 (Bcl-2 homology 3) binding cleft (Zhang et al., 2006). This finding raises the possibility of alternate mechanisms favouring interactions of pro-survival members of the Bcl-2 family. In light of these findings, the concentrations of chelerythrine in our experiments is much lower than the EC₅₀ value for its pro-apoptotic effect (Chan et al., 2003; Maliková et al., 2006). Thus, at low concentrations anti-apoptotic influences of chelerythrine and sanguinarine seem to predominate.

Therefore, our results strongly suggest that the protective function of Hepeel® against cadmium-induced cytotoxicity results from the synergistic actions of its composite tinctures. The decisive anti-apoptotic influence of Hepeel® may be supported by its antioxidative features that help stabilize cellular GSH content, and consequently the sulfhydryl status of cellular proteins. Further studies are needed to discern whether this protective effect is specific to cadmium toxicity in hepatocytes, or can be generalised to other toxins and cell populations.

In conclusion, Hepeel® efficiently antagonised cytotoxic and apoptotic effects of the heavy metal cadmium in hepatocyte cell populations. This protective function is likely based on anti-apoptotic influence distinct from anti-oxidative function, but may be rendered more efficient by the synergistic effects of both. These observations add to the list of beneficial effects recently reported with this preparation (Gebhardt, 2003), and support the possible therapeutic use of Hepeel®, particularly for cases of heavy metal poisoning.

Conflict of interest

None.

Acknowledgement

This work was supported in part by the University of Leipzig (KST 764100100); and Biologische Heilmittel Heel GmbH, Baden-Baden, Germany (97000-050). The author would like to thank Mrs. D. Keller, Mr. F. Struck and Mrs. B. Woite for excellent technical assistance and Dr. A. Gerasimova for valuable comments and editing.

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