Fria radikaler och antioxidanter

KAPITEL 1
Fria radikaler och antioxidanter
Ett omfattande material ingår i ”Den Nya Medicinen”, upplaga II 2008. Här skall
jag i stället redovisa ämnet i en mera vetenskaplig form i tre artiklar på
engelska.
1. Hur modellen utvecklades.
2. Analys av erytrocytskörhet.
3. Behandlingsresultat.
-4-
A MODEL FOR ANTIOXIDANT
TREATMENT OF CHRONIC
DEGENERATIVE DISEASES
PART I. HOW THE MODEL WAS DEVELOPED
DOES CURRENT RESEARCH ON
ANTIOXIDANTS HAVE
SERIOUS FAULTS?
Per-Arne Öckerman
MD, PhD, Emeritus Professor of Clinical Biochemistry
University of Lund, Sweden
Suddeviksvägen 12
S-430 94 Bohus-Björkö
Sweden
-5-
SUMMARY
Free radicals play a role in many chronic degenerative and autoimmune
diseases. In accordance with this knowledge, many studies have been
published of the effect of antioxidant treatment. Absence of a positive effect in
such studies has often been taken as evidence that antioxidants in the form of
supplements are of no value, even deleterious.
Taking into consideration own bed-side observations in patients as well as
known facts of mechanisms of action for antioxidants a method was developed
for assay of free radical activity, suitable for use in clinical practice and an
effective antioxidant treatment program was designed. This program, involving
control by free radical assay that increased oxidative stress is successfully
diminished by a broad spectrum of high-dose antioxidants, has been used as
basis in controlled studies and to monitor treatment in the individual patient.
Highly positive treatment results will later be described in many chronic
diseases.
It is suggested that this may indicate a possibility to change medicine, both by
more effective therapy and by a more active prevention and that most of the
studies published on treatment with antioxidants must be redone. Future
research must use a broad spectrum of antioxidants and control the effect by
analysis of free radicals.
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INTRODUCTION
When it was understood that free radicals play a role in many chronic
degenerative and autoimmune diseases, great hopes were awakened that
antioxidants could help to prevent and even treat such disorders. Today it is
generally considered that these hopes have not been fulfilled and it is often
stated that antioxidants in preparations are ineffective or even dangerous (1-3)
Antioxidants in food on the other hand have been found to be more active and
this has been explained as an effect of the more complex composition of food
(1,4-5).
Own bed-side observations in patients and considerations of known facts about
mechanism of action of antioxidant gave motive to reconsider the conclusions
in the literature and find a model for more effective treatment with
antioxidants.
MATERIAL AND METHODS
Patients:
This is a retrospective study of all patients (n= 5000) from my practice in
complementary and alternative medicine (CAM) during the years 1988 – 2005
(October). These patients came asking if there was a CAM treatment for them.
They had all been thoroughly examined medically in the established system,
but were not satisfied with diagnosis, lack of diagnosis or treatment
propositions or results. There was a very broad spectrum of diagnoses among
these patients.
All patients were analyzed for free radical activity. If an increased activity was
found, they were offered antioxidant treatment with repeated assays of free
radical activity as control of the effect of treatment. Results presented here
originate from 2400 patients, who have had a full antioxidant treatment and
free radical assay as well as adequate clinical control during 1995 -2005.
Analyses:
Assay of free radicals was performed using the following methods:
1. 8-OH-deoxyguanosine in urine (6)
2. Malondialdehyde in urine (7)
3. Micronucleus induction in erythrocytes (8)
4. FRAS 3 (9)
5. Erythrocyte fragility test (10-11).
Simple facts about the methods: Method 1 measures damage to DNA and can
be said to be a reference method with which other methods can be compared.
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Method 2 assays peroxidation to lipids. Method 3 estimates fragmentation of
nuclear materal. Methods 1-3 are not suited for routine clinical use, while
Methods 4 and 5 are easily applicable in clinical practice. Method 4 evaluates
coloured products of free radical reactions.
Method 5 is based on the following principle: An excess of free radicals
peroxidizes lipids in cell membranes, including erythrocytes. When the cells are
exposed to air on a glass slide they are haemolysed, which can be seen in a
microscope at low magnification as white “lakes”. The area of the “lakes” is
used on a scale from 0 to 50 as an estimate of damage by free radicals. This
method is further described in DISCUSSION and in (10-11).
Fig. 1 A-B. Erythrocyte fragility test
-8-
The Erythrocyte fragility test is easily applicable in clinical practice, is extremely
sensitive, and could, in fact, easily be used at any patient visit to a practician as
well as a research tool.
Clinical evaluation was performed with normal procedures according to
diagnosis, when available taken from specialist reports.
RESULTS
Treatment.
How a model was developed.
Bed-side observations: When I started to make analysis of free radicals in my
patients in 1988, I noticed several patients who were taking what was then
considered high doses of antioxidants (e.g. selenium, 200 ug, vitamin E, 400 mg
-9-
and vitamin C, 1 g). Yet, they did not feel well and came to me, hoping to get a
more effective treatment. I noticed a high degree of damage in the erythrocyte
fragility test and gave them a broader spectrum of antioxidants with effect
both on their symptoms and on the analysis.
Theoretical considerations: I studied the literature, talked to colleagues with
experience of antioxidants and, not least, learnt from the results in my patients
and started to compose a model for effective antioxidant treatment. This
model is still developing. The principles can be described thus:
Model used.
1. Give all vitamins, trace elements and minerals in recommended daily
intake doses (RDI). This is because it is impossible by analysis to exclude
all deficiencies and in order to avoid imbalance and deleterious effects
that can be caused by giving high dose of single components. Iron is
given only when a deficiency has been proven, since iron in excess
counteracts the anti-free radical effect.
2. Give enough magnesium to counteract the overacidity that exists in most
patients.
3. Give much higher doses of the B-vitamins and of the antioxidants vitamin
C and E and selenium.
4. Give a broad spectrum of natural non-vitamin antioxidants: lycopene,
citrus bioflavonoids and pollen extract.
5. The program must be effective in most patients in 1-3 months.
6. It must not be more complicated or more expensive than absolutely
necessary.
Practical aspects.
This is how I give antioxidants to patients:
1. All normal medical procedures including diagnosis and treatment must
first have been performed.
2. Assay of oxidative stress has demonstrated an excess activity of free
radicals. When no such excess is present, no antioxidant treatment is
given.
3. Effective antioxidant treatment is given in the dose needed until
maximum effect is obtained. Effect is measured as decrease of free
radical activity and clinical symptoms and signs.
4. Antioxidants are given for as long as needed at the minimum dose that
has full effect.
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A positive effect on clinical symptoms and on erythrocyte fragility test is usually
seen after one to three months, sometimes even earlier, in severe cases it may
take longer.
Treatment must in most cases continue for a very long time. If in doubt
whether treatment is really effective or whether the dose used is necessary,
stop treatment or decrease dose. In my experience symptoms will usually
reappear within one week, if antioxidants are still needed.
DISCUSSION
Erythrocyte fragility test.
It has been demonstrated that oxidative stress induces haemolysis of
erythrocytes(12-14) by decreasing surface charge density(14) and that
antioxidants can restore erythrocyte membrane integrity (12,14).
In the Erythrocyte fragility test the cells are exposed to air, which involves a
strong oxidative stress to the erythrocytes. Erythrocytes that have already been
damaged in vivo by an excess of free radicals would be more prone to
haemolysis than intact cells. In the method used such haemolysis is seen as
lacunae in the microscope at low magnification. The fact that there is a
concordance between positive clinical effect and a decrease in the erythrocyte
fragility test, as demonstrated in the following parts in this series adds
credibility to the use of this test.
Meta-analysis in The Lancet.
In a recent and very much discussed article (15), a compilation of many
scientific studies of antioxidants has been published. The following conclusions
have been drawn in this meta-analysis.
1. Antioxidants do not protect against gastro-intestinal cancer.
2. Antioxidants might even cause premature death.
- 11 -
In my opinion, these conclusions are correct, but the authors and even more
so, the media, do not follow normal logics. What has been shown is actually:
1. One, two or three antioxidants have no protective effect against gastrointestinal cancer.
2. High doses of one, two or three antioxidants may even be dangerous.
But, when it is stated, that antioxidants have no positive effect, the authors do
not follow normal logical rules: You can never prove that a thing does not exist.
It can quite reasonably be a fact that all studies referred to studied antioxidants
in the wrong way. The study might only demonstrate how not to perform
studies on antioxidant effects in patients.
Principles for antioxidant treatment.
The purpose of keeping free radical damage at bay is to avoid all such factors
that increase production of free radicals, e.g. smoking, toxins, too much food or
too much exercise, infections, etc. and to get enough antioxidants through food
or food supplements.
Antioxidant treatment is indicated in any situation, where an increased activity
of free radicals has been found by blood test. In practice this can occur in nondiseased individuals, as well as in patients with a very large number of different
diseases, e.g. all chronic degenerative diseases, all autoimmune diseases,
patients treated by cytostatics, and so on. The important thing here is to
understand that consideration of free radical damage has very little to do with
consideration of a diagnosis of a “specific” disease.
The antioxidant conflict.
It has long been pointed out that oxidative stress is of importance in many
diseases, but antioxidant treatment has in most cases been of much less value
than hoped for. Many doctors, therefore, to-day state that buying antioxidants
is to throw your money away.
In my opinion, most scientific studies have been performed in a way that does
not take into consideration important known facts. Because of this
unmotivated conclusions have been drawn.
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1. Most studies have used only one or a few antioxidants. However, one
single antioxidant cannot solve the problem. Many antioxidants are
needed in order to get full protection against all the different free
radicals.
2. The doses have been too low.
3. In any study of antioxidant effects, analysis must be performed of
oxidative stress (free radical activity). This has not been done. If the
included patients did not have increased oxidative stress or did not
during treatment obtain normalised values for free radicals, the
treatment cannot be evaluated. In the meta-study (15) this is actually
mentioned as a weakness.
- 13 -
REFERENCES
1. Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL;
Nutrition Committee of the American Heart Association Council on
Nutrition, Physical Activity, and Metabolism. Antioxidant vitamin
supplements and cardiovascular disease. Circulation 2004;110(5):637-41.
2. Lee IM, Cook NR, Manson JE, Buring JE, Hennekens CH. Beta-carotene
supplementation and incidence of cancer and cardiovascular disease: the
Women´s Health Study. J Natl Cancer Inst 1999; 91(24): 2102-6.
3. Ascherio A, Rimm EB, Hernan MA, Giuvannucci E, Kawachi I, Stampfer
MJ,Willett WC. Relation of consumption of vitamin E, vitamin C, and
carotenoids to risk for stroke among men in the United States. Ann Intern
Med 1999;130(12): 963-70.
4. Joshipura KJ, Hu FB, Manson JE, Stampfer MJ, Rimm EB, Speizer FE,
Colditz G, Ascherio A, Rosner B, Spiegelman D, Willett WC. The effect of
fruit and vegetable intake on risk for coronary heart disease. Ann Intern
Med 2001; 134(12): 1106-14.
5. Joshipura KJ, Ascherio A, Manson JE, Stampfer MJ, Rimm EB, Speizer FE,
Hennekens CH, Spiegelman D, Willett WC. Fruit and vegetable intake in
relation to risk of ischemic stroke. JAMA 1999;282(13): 1233-9.
6. Tagesson C, Källberg M, Leanderson P. Determination of urinary 8hydroxydeoxyguanosine by coupled-column high-performance liquid
chromatography with electrochemical detection: a non-invasive assay for
in vivo oxidative DNA damage in humans. Toxicol Methods 1992;1: 24251.
7. Tagesson C, Källberg M, Wingren G. Urinary malondialdehyde and 8hydroxydeoxyguanosine as potential markers of oxidative stress in
industrial art glass workers. Int Arch OccupEnviron Health 1996;69: 5-13.
8. Abramsson-Zetterberg L, Zetterberg G, Bergqvist M and Grawé J. Human
cytogenetic biomonitoring using flow-cytometric analysis of micronuclei
in transferrin-positive immature peripheral blood reticulocytes. Environ
Mol Mutagen 2000; 36: 22-31.
9. Franzini A, Luraschi P, Pagani A. Direct measurement of reactive oxygen
metabolites in blood serum: analytical assessment of a novel method.
Biochimica Clinica 1996; Suppl.1/5-6:89.
10. Öckerman PA. Free radicals in electromagnetic hypersensitivity: A simple
and sensitive method for assay of damage to erythrocytes caused by free
radicals. Clinical Practice of Alternative Medicine 2000; 1: 81-87.
- 14 -
11. Öckerman PA. A model for antioxidant treatment of chronic
degenerative diseases. Part II. Erythrocyte fragility test. Interpretation of
microscopic slides. eCAM 2006…..
12. Gupta V, Gupta A, Saggu S, Divekar HM, Grover SK, Kumar R. Anti-stress
and adaptogenic activity of L-arginine supplementation. eCAM 2005;
2(1): 93-97.
13. Bureau A, Lahet JJ, Lenfant F, Bouyer F, Petitjean M, Chaillot B, Freysz M.
Optimization of a model of red blood cells for the study of anti-oxidant
drugs, in terms of concentration of oxidant and phosphate buffer.
Biomed Pharmacother 2005; 59(7): 341-4.
14. Sangeetha P, Balu M, Haripriya D, Panneerselvam C. Age associated
changes in erythrocyte membrane surface charge: Modulatory role of
grape seed proanthocyanidins. Exp Gerontol 2005;40(10): 820-8.
15. Bjelakovic G, Nikolova D, Simonetti RG and Gluud C. Antioxidant
supplements for prevention of gastrointestinal cancers: a systematic
review and meta-analysis. Lancet 2004; 364: 1219-1228.
- 15 -
A MODEL FOR ANTIOXIDANT
TREATMENT OF CHRONIC
DEGENERATIVE DISEASES
PART II. ERYTHROCYTE FRAGILITY TEST
INTERPRETATION OF
MICROSCOPIC SLIDES
Per-Arne Öckerman
MD, PhD, Emeritus Professor of Clinical Biochemistry,
University of Lund, Sweden
Suddeviksvägen 12
S-430 94 Bohus-Björkö
Sweden
- 16 -
SUMMARY
Assaying damage to erythrocytes by free radicals in the Erythrocyte fragility
test involves interpretation of microscopic slides. How this is performed is
described, including requirements on the properties of the sample, artefacts
and method of score calculation.
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SAMPLING AND INTERPRETATION
Capillary blood is taken by a special technique from a finger. The sample can be
analysed after 5-10 minutes in the microscope. The number of “lakes” is
assayed and shown directly to the patient on a TV monitor. This means that
there is extremely good communication between doctor and patient. The
patient will really understand what the test means. If the doctor cannot
interpret the sample, it can be sent by post to a laboratory for analysis.
This assay is very well suited for an appraisal of the amount of excess free
radical activity in the individual at each time. I think this test should be
performed at every patient visit to a doctor. Any increased value motivates a
discussion of cause and antioxidant treatment.
As described below, five drops are put on the slide from one drop on the
fingertip. Each drop on the slide is given a figure for signs of damage (“lakes”)
from 0 – 10 according to the scale presented in Fig. 1-31. The figures for all five
drops are added to obtain a scale from 0-50 arbitrary units. Most healthy
individuals demonstrate measurable damage with this method, having values
between 0 and 15. Any value above 15 can indicate a problem and an
indication for discussing cause and antioxidant treatment.
This method also is well suited for controlling the effect of treatment.
Erythrocyte fragility test demands skill in capillary blood sampling and in
interpreting the slides in the microscope.
Erythrocyte fragility test
Sampling technique
Material:
A clean and dry glass slide with frosted part for name.
Cleaning solution and wad.
Dry wad.
Lancet for sampling of capillary blood. Must be small and round,
not triangular (low-flow type). The usual lancets (normal-flow type) give
an incision that is too big.
Gloves to avoid infection risk for the sampler.
- 18 -
Sampling:
1.
2.
3.
4.
5.
Prepare one glass slide by writing patient´s name and sampling date.
Sit directly opposite to the patient.
Clean the finger-tip (finger III or IV).
Dry with a clean wad.
Take the patient´s finger between your own thumb and index finger.
Stretch the skin slightly.
6. Make a small incision at the side of the finger-tip. The incision should be
smaller than usual.
7. Press slightly to obtain a drop. Wipe off this first drop. Press slightly to
obtain a new drop. Let this drop stay for 15 – 20 seconds. It must not
increase in size. Thus this is different from the normal sampling
technique, where free flow is important.
8. After 15 – 20 seconds press the glass lightly against the drop of blood five
times in rapid succession. See to it that the drops and the patient´s name
come on the same side of the glass. The glass must be parallel to the skin
surface. The glass should just touch the skin. If you only touch the drop
of blood, but not the skin, the drop on the glass will become too thick. If
you press too hard against the skin, the drop on the glass will be too thin
with uneven circumference.
9. Put the slide on an horizontal surface, until all the drops are completely
dry.
10.Then, look at all the drops. None must be too thick (seen as a red, raised
circle at the border with less color in the middle). The last drops must not
be too small or be flowing out.
11.Control that you have written name and sampling date on the slide.
12.The slide must be protected from mechanical damage by being kept in a
glass container. It can be sent by post in a small plastic container. It can
be kept for several years.
In order to be able to interpret the slides with blood samples a detailed
knowledge of what can be seen in the microscope is necessary.
- 19 -
Fig. 1
Fig. 2
Drop is much too thick. Cannot be interpreted.
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Fig. 3
This drop is too thick in the periphery. ”Lakes” just inside the peripheral ring are artifacts.
This drop cannot be used.
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Fig. 4
Same as last picture, but slightly less evident.
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Fig. 5
This drop is a little bit too small. Can be interpreted.
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Fig. 6
Small drop.
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Fig. 7
Very small drop. Do not use for interpretation of free radicals.
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Fig. 8
Technically OK. Artefact in the form of a fibre. Do not use the area near the fibre for
interpretation.
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Fig. 9
Artefact in the form of a small grain.
- 27 -
Fig. 10
This drop has been squeezed. The ”lakes” in the squeezed area are artefacts. Do not
interpret.
- 28 -
Fig. 11
Scale for free radicals (FR). This is FR=0.
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Fig. 12
FR=1.
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Fig. 13
FR=2.
- 31 -
Fig. 14
FR=3.
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Fig. 15
FR=4.
- 33 -
Fig. 16
FR=5.
- 34 -
Fig. 17
FR=6.
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Fig. 18
FR=7.
- 36 -
Fig. 19
FR=8. Too thick with a peripheral ring. However, the lakes are not only just inside the ring,
but are evenly distributed (and there is the same picture in the following drops, see next
slide).
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Fig. 20
FR=8. Slide 2 from the same patient as in the previous slide.
- 38 -
Fig. 21
FR=9.
- 39 -
Fig. 22
FR=10.
- 40 -
Fig. 23
Indicates intestinal toxicity or detoxication work in the liver.
- 41 -
Fig. 24
Same as previous.
- 42 -
Fig. 25
A larger black area in the middle than normally. Indicates toxicity.
Radial elongated lakes called spokes. Indicates calcium imbalance (Serum calcium is normal).
- 43 -
Fig. 26
The large number of small, round or oval lakes in the middle indicate mental stress. Normally
seen in drops 2-3.
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Fig. 27
Parts with less red colour. Smaller degree of free radical damage.
- 45 -
Fig. 28
Black star-like figures thought to indicate some kind of hormonal disturbance, unclear what.
- 46 -
Fig. 29
Uneven pattern of the fibrin. Importance not clear.
- 47 -
Fig. 30
Very small round lakes seen in all small drops. Possibly allergy-indicator.
- 48 -
Fig. 31
Brownish colour. Often seen together with spokes. Do not use such drops for interpretation of
free radicals.
REFERENCE
1. Öckerman PA. Free radicals in electromagnetic hypersensitivity: A simple
and sensitive method for assay of damage to erythrocytes caused by free
radicals.
Clinical Practice of Alternative medicine 2000; 1(2):81-87.
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A MODEL FOR ANTIOXIDANT
TREATMENT OF CHRONIC
DEGENERATIVE DISEASES
PART III.
POSITIVE EFFECT OF POLLEN EXTRACTS IN THE
TREATMENT OF CHRONIC FATIGUE SYNDROME
Per-Arne Öckerman
MD, PhD, Emeritus Professor of Clinical Biochemistry
University of Lund, Sweden
Suddeviksvägen 12
S-430 94 Bohus-Björkö
Sweden
- 50 -
SUMMARY
Groups of patients with chronic fatigue syndrome were treated for one month
with three different preparations of pollen extract. Over-all well-being and
fatigue were measured by self-estimation. Free radical activity was assayed on
erythrocytes as lipid peroxidative damage and as micronucleus induction.
A highly significant reduction of fatigue as well as a highly significant
improvement of over-all well-being was noted with all preparations. Also
damage to erythrocytes by free radicals was significantly decreased in all
treatment groups. No significant changes were seen in the placebo groups.
It is concluded, that, if larger studies can verify the present results, pollen
extracts may help patients with chronic fatigue syndrome, a condition
otherwise considered difficult to treat.
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INTRODUCTION
Chronic fatigue syndrome (CFS) is characterized by persistent, severe fatigue
and numerous other symptoms involving CNS, intestines, muscles and other
systems. The etiology of CFS remains unclear; however, recent studies have
indicated that oxidative stress may be involved in its pathogenesis (1-3). A
variety of treatments have been studied, of which graded exercise and
cognitive behavioural therapy have been considered to show some positive
results (4).
In an earlier controlled, double-blind study it was shown that a pollen extract
had a significant effect on several symptoms in patients with CFS(1). These
results motivated further studies to find out the optimum dosage as well as
possible differences between available pollen extracts.
In this study a comparison has been made between three different pollen
preparations in controlled, double-blind, simulated, single-blind mode as well
as in open studies.
MATERIAL AND METHODS
Patients: All patients had CFS as defined (1,5). This means that they had for at
least six months had a more than 50 per cent decrease of their capacity due to
fatigue, that they had at least six other symptoms from various organ systems
and that thorough clinical investigation had been unable to find any other
disease that could explain their symptoms.
Inclusion criteria: A diagnosis of CFS. Age 20-70. Symptom score 5 or higher
(See below). Erythrocyte fragility score: 15 or higher (See below).
Exclusion criteria: Smoking. Treatment with other antioxidants or drugs.
Analyses: Symptom score was estimated by the patient for energy/fatigue and
quality of life as described in SF-36 Health Survey (6). This system has been
demonstrated to be applicable to Swedish patients (6). The scale was inverted
compared to SF-36 and simplified to go from 0 (=no symptoms) to 10
(=extremely severe symptoms) (1). The patients were asked to give a figure
from 0 to 10 describing their own estimation of how tired they were (fatigue)
- 52 -
and how they rated their health problems (quality of life, here called total wellbeing or over-all symptoms). It is not possible to describe exactly the meaning
of these figures, but any change of a figure during treatment should mirror a
change in self-estimated symptoms.
Erythrocyte fragility test was used as an indicator of an excess of damage from
free radicals on red blood cell membranes, on a scale from 0 (=no damage
visible on erythrocytes) to 50 (extreme damage) (1,7). This method shows
acute increase of free radical activity within a few days. However, when free
radical activity is counteracted by effective antioxidant treatment, damaged
erythrocytes are not healed, but only disappear in accordance with their
normal life-span of four months. Thus, this method can be used to demonstrate
short-time changes by increased free radical activity (7). For studies of
antioxidant effects treatment time must be longer, preferably one month.
More information on this method will be given separately (8).
Damage to nuclei by free radicals was assayed by micronucleus induction in
erythrocytes as described by Abramsson-Zetterberg and co-workers (9).
Statistics: All comparisons are made by a paired two-sample student´s t-test.
Pollen preparations: Three different pollen preparations were used. All
preparations were produced by Allergon AB, Välinge 2090, SE-262 92
Ängelholm, Sweden. This company has a unique technique for producing
allergen-free pollen extracts. All extracts are produced in a similar way and
contain a slightly variable mixture of water- and lipid-soluble components.
Since all contain a complex mixture, an exact description of “specific”
ingredients is not possible (10).
1. Royal Sport extract. This is a water-soluble extract, distributed by Beauty
Pollen Co.,Ltd., Tokyo, Japan.
2. MIROR SOD. This is a preparation of cracked pollen in tablet form
distributed by MIROR, Blanc Int´l Co. Ltd, Taipei, 10456 Taiwan. It
contains Polbax pollen extract. Polbax is a registered product of waterand lipidsoluble extracts. This is the preparation used in an earlier study
(1). A very low dose of Polbax was used as a simulated placebo at a dose
of 1 % of the dose used in the MIROR SOD-group. This is the same
preparation as used at a 14 times higher dose in an earlier study (1).
3. Mini-G. This is a preparation of cracked pollen in tablet form distributed
by MIROR, Blanc Int´l Co Ltd, Taipei, 10456 Taiwan.
4. Placebo. This preparation did not contain any material from pollen.
- 53 -
Study groups:
Treatment time in all groups was one month.
1. Controlled, double-blind, crossed-over study: Fifteen patients were given
3.2 g of Royal Sport extract or placebo in the form of granules for one
month. After two weeks without treatment (wash-out) they were given
placebo or 3.2 g of Royal Sport extract for one month. Analysis was
made of symptom score and free radical damage to nuclei (9).
2. Open study. Patients were given 3.2 g of Royal Sport. Analysis of
symptom score and erythrocyte fragility. Number of participants
(n)=184.
3. Simulated placebo. A very low dose of Polbax was used as a simulated
placebo. n= 20.
4. Mini-G.. Dose used was 2.4 g per day (12 tablets) for one month. n=20.
5. MIROR SOD. Dose was 1.5 g (2 sachets) per day. n=20.
6. Combination of Mini-G + MIROR SOD. In this part the patients took both
preparations in the same dosage as in 3 and 4. n=20. This thus means
that an even higher dose was used than in groups 4 and 5.
Most of the participants in study groups 3 - 6 were the same. There was a
wash-out time of at least one month, before an individual started in a new
study group. Normally, the effect of treatment tended to disappear within
one week. Study groups 1 and 2 consisted of participants separate from
those in 3-6.
RESULTS
Controlled study. High dose Royal Sport.
In the controlled study (test 1) there was a significant decrease in over-all
symptoms as well as in free radical damage to nuclei (Fig. 1). There were no
significant side-reactions noted.
Fig. 1-18. Change in symptom score and free radical activity. Patients with
Chronic fatigue syndrome were given treatment with three different pollen
extracts as described in Materials and methods. Analysis was made of over-all
condition and fatigue on a self-estimation scale (1) and of free radical damage
to erythrocytes by a microscopic method (8) or by assay of micronucleus
induction in erythrocytes (9). Scale was from 0 (no symptoms)-10 (extreme
- 54 -
symptoms) for over-all condition and fatigue and from 0-50 for free radical
damage to erythrocytes. Values given are mean values (+/-SD) before any
treatment and after each individual treatment.
- 55 -
Royal Sport Controlled Study. (Fig. 1-4)
There was a highly significant decrease of over-all symptoms and a significant
decrease of micro-nuclei in the verum group, but no significant change in the
placebo group.
Fig.1. Royal Sport, verum, over-all symptoms
Royal Sport controlled study
9
8
7
Symptom score
6
5
4
3
2
1
0
2
1
Time
Mean
SD
p
Change
7.85
1.72
6.23
2.55
***
- 21%
- 56 -
Fig.2. Royal Sport, verum, micro-nuclei
Royal Sport Free radicals
1,8
1,6
1,4
Micronuclei
1,2
1
0,8
0,6
0,4
0,2
0
1
2
Time
Mean
SD
p
Change
1.55
0.67
1.11
0.55
*
- 28%
- 57 -
Fig.3. Royal Sport, placebo, over-all symptoms
Royal Sport placebo
8
7
6
Symptom score
5
4
3
2
1
0
1
2
Time
Mean
SD
p
6.63
2.30
6.65
2.27
n.s.
- 58 -
Fig.4. Royal Sport, placebo, micro-nuclei
Poyal Sport placebo
Micronuclei
1
0
1
2
Time
Mean
SD
p
1.43
0.54
1.32
0.50
n.s.
- 59 -
Royal Sport Open Study. (Fig. 5-6)
There was a highly significant decrease of over-all symptoms and in the
erythrocyte fragility test.
Fig.5. Royal Sport, open study, over-all symptoms
Royal Sport open study
8
7
6
Symptom score
5
4
3
2
1
0
1
2
Time
Mean
p
Change
6.80
3.82
***
- 44%
- 60 -
Fig.6. Royal Sport, open study, erythrocyte fragility test
Royal Sport open study
25
Ery-frag. score
20
15
10
5
0
1
2
Time
Mean
p
Change
23.0
10.8
***
- 53%
- 61 -
Simulated placebo. Very low dose Polbax. (Fig.7-9)
There was no significant change during treatment with extremely low-dose
Polbax (test 3). Most patients did not register any change in symptoms. There
were no side effects noticed.
Fig.7. Simulated placebo. Over-all symptoms
Simulated placebo
Symptom score
10
0
1
2
Time
Mean
SD
p
7.23
1.42
7.15
1.34
n.s.
- 62 -
Fig. 8. Simulated placebo. Fatigue
Simulated placebo
Fatigue
10
0
1
2
Time
Mean
SD
p
7.46
1.34
7.15
1.68
n.s.
- 63 -
Fig. 9. Simulated placebo. Erythrocyte fragility test
Simulated placebo
30
Ery-fragility
20
10
0
1
2
Time
Mean
SD
p
24.2
8.36
23.2
8.07
n.s.
- 64 -
Mini-G or MIROR-SOD (Fig. 10-15)
Treatment with Mini-G or MIROR-SOD (tests 4 and 5) highly significantly
diminished both over-all symptom score, fatigue and erythrocyte fragility.
Fig. 10. Mini-G. Over-all symptoms.
Mini-G Over-all symptoms
Symptom score
10
0
1
2
Time
Mean
SD
p
Change
6.92
1.66
4.92
1.93
***
- 29%
- 65 -
Fig. 11. Mini-G. Fatigue
Mini-G. Fatigue.
Fatigue score
10
0
1
2
Time
Mean
SD
p
Change
7.42
1.46
4.68
1.80
***
- 37%
- 66 -
Fig. 12. Mini-G. Erythrocyte fragility test
Mini-G. Ery- fragility
Ery-frag
30
0
1
2
Time
Mean
SD
p
Change
23.7
7.30
14.5
6.78
***
- 39%
- 67 -
Fig. 13. MINOR-SOD. Over-all symptoms
MIROR-SOD symptom score
Symptom score
10
0
1
2
Time
Mean
SD
p
Change
7.71
1.46
5.14
2.28
***
- 33%
- 68 -
Fig. 14. MIROR-SOD Fatigue
MIROR-SOD Fatigue
Fatigue score
10
0
1
2
Time
Mean
SD
p
Change
7.86
1.29
4.86
1.76
***
- 38%
- 69 -
Fig. 15. MIROR-SOD Erythrocyte fragility test
MIROR-SOD Ery-fragility
30
Ery-frag
20
10
0
1
2
Time
Mean
SD
p
Change
23.7
6.93
11.1
7.31
***
- 53%
- 70 -
Combining Mini-G and MIROR-SOD (Fig. 16-18)
Combining both preparations (test 6) gave an even better result with a highly
significant reduction of over-all symptom score, fatigue and erythrocyte
fragility.
Fig. 16. Combining Mini-G and MIROR-SOD. Over-all symptoms
Combination. Symptom score.
10
9
8
Symptom score
7
6
5
4
3
2
1
0
2
1
Time
Mean
SD
p
Change
8.00
0.89
4.57
2.42
***
- 43%
- 71 -
Fig. 17. Combining Mini-G and MIROR-SOD. Fatigue
Combination. Fatigue.
10
9
8
Fatigue score
7
6
5
4
3
2
1
0
1
2
Time
Mean
SD
p
Change
7.71
1.19
4.43
1.63
***
- 43%
- 72 -
Fig. 18. Combining Mini-G and MIROR-SOD. Erythrocyte fragility
Combination Ery-fragility
30
25
Ery-fragilty
20
15
10
5
0
1
2
Time
Mean
SD
p
Change
24.0
6.37
10.6
3.60
***
- 56%
- 73 -
Side-effects
There were no significant side effects in any of the tests. A few of the patients
noticed a difference between the two preparations, preferring either one or
the other. The combined treatment in test 6 gave a similar effect as the
individually best preparation in tests 4 and 5. In some patients an even better
effect was noted in test 6 than in either test 4 or 5.
DISCUSSION
It has earlier been demonstrated that pollen extracts contain a broad spectrum
of highly active antioxidants (1, 10-11).
In this study further proof is presented that pollen extracts do, indeed, give a
significant reduction of symptoms, when damage by free radicals on
erythrocyte membranes or reticulocyte nuclei is reduced.
It is generally accepted that an excess of free radicals gives rise to oxidation of
DNA and peroxidation of unsaturated fats in cell membranes. This damage
would lead to problems with cell and cellular organelle functions. Possibly,
improvement of such functional damage may explain the findings in this and
earlier studies in this series (1, 11).
Comparison between the preparations: All preparations gave significant
improvement of symptoms and reduction of damage to red blood cells caused
by free radicals, measured with two different methods.
In the semi - open studies slightly better results were obtained as compared to
the controlled, double blind studies (this and my earlier paper (1)).
A comparison between all tests included in this paper and in ref.1
demonstrates that the tested extracts are highly effective. Dose and treatment
time are of importance: High dose and treatment time until effect is obtained
may be needed for full effect.
The highly significant effect demonstrated here indicates that pollen extracts
after only one month of treatment can help patients with a chronic disease,
earlier considered very difficult to treat. Of course, this has to be verified in
larger, controlled studies.
- 74 -
Since CFS is a common condition, such larger studies of treatment with pollen
preparations can be considered indicated.
ACKNOWLEDGEMENTS
The controlled, double-blind part of these studies was accepted by the
Research Ethical Committee of Göteborg University.
Dr. Jan Grawé, Stockholm University, performed the assays of micronucleus
induction in erythrocytes.
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