2009 - The effect of KAQUN-water on the

immune parameters of healthy volunteers

/NICS/

NATIONAL INSTITUTE OF CHEMICAL SAFETY

 Report

The effect of KAQUN-water on the immune parameters of healthy volunteers

Budapest

2009

Report
The effect of KAQUN-water on the immune parameters of healthy volunteers

Antecedents

KAQUN HUNGÁRIA Ltd. (2144 Kerepes, Szabadság út 102), as Client has contracted the
National Institute of Chemical Safety/NICS) (1096 Budapest, Nagyvárad tér 2.) as contractor
in contract no. GOKBI-360/2009 to test the immune effects of KAQUN water = Q voda in
healthy volunteers at the Department of Cytogenetics and Immunology of NICS. Kaqun is a
special water with high oxygen content, suitable for use in the form of drinking water or
bathing. In our study we examined the effect of 21 days of bathing and drinking on the
immune parameters of healthy volunteers. The end points measured were: qualitative and
quantitative blood counts, the ratio of lymphocyte populations, lymphocyte activation and
the oxidative burst of neutrophil granulocytes. The measurements were carried out on the
first day before the start of the treatment (0 point) and on the 8 th, 15th and 21st days.

The theoretical basis of immunology tests

Immune-toxicology examines the damaging/modifying effects caused by exposure at the

workplace, environment or therapy on the immune system. Its task is to detect and assess
the modifying factors affecting the immune system especially from the aspect of their effect
on human health. An immune response may be elicited when the immune system is the
passive target of a chemical agent or when the chemical, as an antigen, triggers a specific
response. In consequence of the complexity of the immune system the chemical agents have
a broad target of attack. They can affect the development, maturation, division,
differentiation and function of cells, or modify the regulation of the immune system.

The immunology tests were carried out on peripheral blood samples. Blood cells consist of
red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes).
The volume ratio of blood cells in the blood is characterized by the hematocrit value.

Erythrocytes are formed in the bone marrow, their development takes about 4 -5 days, while
their nucleic acid content gradually degrades, and mature red blood cells do not have a
nucleus. The blood of an adult contains an average of 4.5x10 12 / l erythrocytes (for women
the average is about 4,5x1012 / l, for men it is somewhat higher, 5x1012 / l). During
maturation erythrocytes synthesize hemoglobin molecules, which are able to carry oxygen.
The average life-span of erythrocytes is 120 days, and they are degraded in the spleen and
liver. More than 99% of the blood cells are erythrocytes.

White blood cells have an important role in the defence mechanisms of the body. Blood
contains an average of 9x109 / l white blood cells, but 4-10 x 109 / l is also within the normal
range. There are 3 main types of leukocytes: granulocytes, monocytes and lymphocytes. 50-
75 % of leukocytes of a healthy person are granulocytes, 20-45 % are lymphocytes and 2-9 %
are monocytes.

The horseshoe shaped nucleus of immature granulocytes becomes lobed as they mature.
Another characteristic feature is the presence of large quantities of granules in the
cytoplasm – the biologically active material stored within them has a very important role in
the development of inflammation and allergic reactions. The neutrophil, basophil and
eosinophil granulocytes can be distinguished on the basis of their histological staining
properties. Most of the granulocytes are neutrophils (3-6 x 109 / l). Since their half life in the
circulation is short, (generally ~6 hours), they are produced in large quantities every day.
They are the basis of cellular protection against infection, and can enter the tissues in large
quantities. In the course of bacterial or fungal infection the neutrophil granulocytes
phagocytose and destroy the pathogens. The intracellular killing of pathogens is achieved by
oxygen-independent enzymes (lysosomal elastase, lysosime) and oxygen-dependent
enzymatic systems (principally NADPH-oxidase). The activated phagocytic cells produce
antimicrobial reactive radicals, so called reactive oxygen intermediates (ROI) in a reaction
named oxidative burst.

Under normal conditions the number of eosinophils is far less in the circulation (1.5-3.0 x 108
/l); they are mostly found in the mucous membranes of the respiratory, urinary, and
intestinal tract participating in the protection against parasites. The number of eosinophils
circulating in the vascular system increases in the case of allergic reactions. The basophils,
similarly to mast cells, contain heparin, histamine and other inflammatory mediators in their
granules. Their number is low (<1 x 108 /l), they are important because they mediate
immediate type hypersensitivity and anaphylactic reactions.

Normally the lymphocyte count is in the range of 1.5-3.5 x 109 /l, and their importance lies in
mediating the adaptive immune response. They are relatively small cells, their round shaped
nucleus fills the cytoplasm almost completely. Lymphocytes are classified into 3 main
groups: T lymphocytes are responsible for the so called cellular immune response, while B
lymphocytes are responsible for the humoral immune response, and the production of
antibodies. The NK cells kill virus infected or cancerous cells.

Monocytes make up about 2-9 % of the white blood cells (1-8 x 108 /l), their nucleus is large,
kidney or bean shaped. They originate from the bone marrow, they then enter the
circulation where they spend about 72 hours, and then pass through the blood vessel wall
and change into tissue macrophages. Their activation is initiated by lymphokines secreted by
T lymphocytes, and as a result they become able to phagocytose foreign matter such as
bacteria, and to release a number of inflammatory mediators (e.g. prostaglandin-E).

Platelets (thrombocytes) are cytoplasmic fragments of megakaryocytes surrounded by a cell-

membrane; they do not have a nucleus. Their size is approximately 2-
the bloodstream or encountering damaged endothelial walls they are activated and play an
important role in blood coagulation. The average thrombocyte count is 3 x 10 11 /l, but a
value in the range of 1.5–4.0 x 1011 /l is normal.

The immune system has an evolutionarily old, non-specific arm which reacts immediately
upon infection. Its most important elements are macrophages, granulocytes, NK cells and
the complement system. Macrophages and granulocytes have an important role in the
phagocytosis of pathogens and foreign particles, while NK cells destroy virus-infected and
cancerous cells. The pathogen organisms that enter the body first meet this so-called innate
immune system. Built on this, is the specific (antigen specific) adaptive immune system,
which reacts slowly (in days) when first meeting the antigen, but has an immunologic
memory; therefore it works fast and efficiently in the case of a second infection. T and B
lymphocytes are the cells of the adaptive immune system. During the adaptive immune
response cytotoxic T (Tc) cells are generated which are able to destroy the pathogens
directly (cellular immune response), and B lymphocytes, which produce antibodies (humoral
immune reaction). The presence of helper T lymphocytes (Th) is essential for the division and
differentiation of the T and B cells. Cell-cell interactions and cytokines produced by
leukocytes have an important role in the regulation of the immune response.

A number of molecules, "markers" appear on the surface of lymphocytes and with their help
the lymphocyte populations can be distinguished from each other. These markers have been
classified into groups, and each marker has been given a CD (Cluster of Differentiation)
number. The basic lymphocyte populations (T, B, NK cells) can be defined with cell markers:
T lymphocytes express CD3 (CD3+ cells), helper T cells also express CD4 (CD4+/CD3+ cells),
cytotoxic T cells express CD8 besides CD3 (CD8+/CD3+ cells). Immature T cells express both
the CD4, and the CD8 molecules (CD4+/CD8+ cells). B lymphocytes can be characterized by
the CD19 cell surface antigen (CD19+cells). NK cells have CD56 surface molecules, but do not
express CD3, therefore they are characterized as CD56+/CD3- cells. CD25 (IL-2 receptor) and
CD71 (transferrin receptor) surface antigens cannot be detected on resting lymphocytes,
they are expressed when the lymphocytes are activated (e.g. by an antigen). Therefore these
surface molecules can be used to detect the activation of lymphocytes.

Immunotoxic materials can affect different immune parameters; therefore we have adjusted
our measurements to characterize different functions. This is important, because the change
in one parameter or another is not suitable to characterize the general condition of the
immune system, conclusions can only be drawn from changes in the data pattern. We
characterized the immune status of the studied subjects by measuring characteristics of
white blood cells gained from peripheral blood. Qualitative and quantitative blood count
was determined, and immune phenotyping was used to determine lymphocyte
subpopulations and the CD25 (IL-2R) and CD71 (transferrin receptor) activation antigens
expressed on lymphocytes with the aid of monoclonal antibodies produced against cell
surface molecules.

Innate immunity was characterized with the help of a functional test: the killing capacity of
white blood cells was determined by measuring the production of reactive oxygen
intermediates (ROI) of granulocytes.
Test procedure

Selection of healthy volunteers

The selection of 30 healthy volunteers (15 women, 15 men) was carried out by KAQUN
HUNGÁRIA Kft. Exclusion criteria in this study were: acute or chronic illness, infection, the
use of any kind of drugs, and smoking, because these could affect immune parameters.
The participants were informed about the purpose and the course of the study, and they
signed a Declaration of Agreement confirming that they had received information about the
study and that their participation was voluntary.

Duration of the study and the procedure:

The examined persons participated in a 21 day bathing and water drinking treatment. The
participants bathed once a day in the morning in individual bathtubs filled with 37 oC water
containing stable oxygen, for a maximum of 50 minutes per occasion. The water drinking
cure consisted of drinking 1.5 liter Kaqun drinking water every day in parallel with the baths.
The bathing cure followed the standards established in the Kaqun Health Program Service.

The 21 days Kaqun bathing and the parallel water drinking treatment was divided into 4
groups, because only 7-8 persons could be examined in a single day. All four groups started
on the first week, the first on Monday, the second on Tuesday, the third on Wednesday and
the fourth on Thursday. The participants of the first group were always examined on
Monday, the second on Tuesday and so on, see table below.

1st week 2nd week 3d week 4th week

day 1 blood day 8 blood day 15 blood day 21 blood

sampling before sampling after sampling after sampling after
Monday 1st group treatment treatment treatment treatment

day 1 blood day 8 blood day 15 blood day 21 blood

sampling before sampling after sampling after sampling after
Tuesday 2nd group treatment treatment treatment treatment

day 1 blood day 8 blood day 15 blood day 21 blood

sampling before sampling after sampling after sampling after
Wednesday 3d group treatment treatment treatment treatment

day 1 blood day 8 blood day 15 blood day 21 blood

sampling before sampling after sampling after sampling after
th
Thursday 4 group treatment treatment treatment treatment
Methods:

Blood sampling:
Blood sampling at the site: day 1 before the bath, (0-point), then on days 8, 15, and 21 after
the bath during the same part of the day. The blood samples were taken from the cubital
vein of the examined persons in sitting position, under sterile conditions with venipunction.
Standard 3 ml sterile vacuum blood sampling tubes containing anti-coagulant were used for
blood sampling. One 3 ml tube with EDTA anti-coagulant for determining the qualitative and
quantitative blood count, one 3 ml tube with heparin for the immunology tests. The blood
samples were given unique identifiers marked on the blood sampling tubes.

The following tests were carried out on the blood samples:

1) Qualitative and quantitative blood count

The qualitative and quantitative blood count was carried out with an automated analyser in
the blood sampling laboratory of OMFI (Bp. IX. Nagyvárad tér 2.).

Determined parameters:

- WBC leukocyte count,

- abs LY, abs MO, abs NEUTR, abs EO: the absolute number of lymphocytes, monocytes,
neutrophil- and eosinophil granulocytes

- LY %, MO %, NEUTR %, EO %, BA %: percentile distribution of lymphocytes, monocytes,

neutrophil- eosinophil- and basophil granulocytes

- RBC red blood cell count,

- Hb concentration of hemoglobin in the blood,

- HTK hematocrit,
- MCV mean cell volume,
- MCHC mean corpuscular hemoglobin concentration,
- RDW-CV red blood cell distribution width
- MCH mean cell hemoglobin,

- Thrombocyte count

2) Determination of immune parameters

Method:

The subpopulations and activation of circulating lymphocytes were determined by immune

phenotyping, using flow cytometry. Heparinized whole blood was used for the
measurement. The surface markers of peripheral lymphocytes were measured with
fluorescent labelled monoclonal antibodies in a flow cytofluorimeter. The surface antigens
examined were: CD3 (T-cell receptor), CD4 and CD8 (T-cell co-receptors), CD19 (B-cell co-
receptor), CD25 (interleukin-2 receptor), CD45 (protein-tirosine-phosphatase, pan leukocyte
marker), CD56 (neural cell adhesion molecule, NK-cell marker), CD71 (transferrin receptor).
Using 3 and 4 colour staining the following antibody combinations were used: (1) CD25-FITC
/ CD8-PE / CD3-PerCP / CD4-APC; (2) CD56-FITC / CD3-PerCP / CD45-APC; and (3) CD71-FITC
/ CD3-PerCP / CD19-APC. Standard forward and side scatter gating combined with CD45 was
used to separate leukocyte populations and to set the lymphocyte gate. The lymphocyte
subpopulations of the donors (T lymphocyte, helper T, cytotoxic T, B lymphocyte and NK-
cell) were determined with the aid of cell markers. CD25 and CD71 surface antigens were
used to determine the activation of lymphocytes.

Determined parameters:

- Ly, Mo, Neu, Eos: percentage of lymphocytes, monocytes, neutrophil- and eosinophil
granulocytes

- Total T, T helper, T cytotoxic, Immature T, B cell, NK-cell: percentage of T lymphocytes,

cytotoxic and helper T lymphocytes, immature T lymphocytes, B lymphocytes and NK-cells
within lymphocytes

- Th/Tc: The ratio of helper and cytotoxic T lymphocytes

- Activated T: percentage of CD25 (IL-2 receptor) activation antigen carrying T cells within the
T cells
- Activated Th: percentage of CD25 activation antigen molecule carrying helper T cells
within the helper T cells
- Activated Tc: percentage of CD25 activation antigen expressing cytotoxic T lymphocytes
within the cytotoxic T lymphocytes
- CD71 positive T: percentage of CD71 (transferrin receptor) molecule carrying T cells
within the T cells
- CD71 positive B: percentage of CD71 (transferrin receptor) molecule carrying B cells
within the B cells
3) Oxidative burst of neutrophil granulocytes

The production of reactive oxygen intermediates (ROI) which is directly proportional with
the killing potential of white blood cells was measured with the aid of Bursttest
(Phagoburst®) kit. Neutrophil granulocytes respond to activation by producing reactive
oxygen intermediates, which oxidize the fluorogenic substrate. The quantity of oxidized
substrate is proportional to the production of reactive oxygen radicals. Heparinized whole
blood was used, and the measurement was carried out on a flow cytometer. We measured
the quantity of oxidized substrate in the control and the stimulated samples, and
determined the percentage of ROI producing cells. The activation stimuli: 1) fMLP
chemotactic peptide (weak stimulus). 2) E. coli opsonized with antibody, which stimulates
through the Fc receptors that recognize the constant part of the antibody (particulate
stimulus) 3) PMA (phorbol-myristil-acetate), which transports signals through protein kinase
C (strong stimulus)

Determined parameters:

Production of reactive oxygen intermediates (ROI)

Control, fMLP, E. coli, PMA: ROI production in unstimulated samples, and samples
stimulated with fMLP, E. coli, and PMA

Percent of ROI producing cells

Control, fMLP, E. coli, PMA: Percent of ROI producing cells in unstimulated samples, and
samples stimulated with fMLP, E. coli, and PMA

Statistical analysis:

Student’s paired-t test was used for the group level statistical evaluation of the results, the
level of significance was set at p<0.05.
 Results and conclusions

1) Qualitative and quantitative blood count

The group results of qualitative and quantitative blood counts are shown in table 1, the
individual results in table 2. No significant change was observed for the group average of
white blood cell count in any of the groups. Individually both increased and decreased
leukocyte counts could be observed during the three weeks of the study. No change was
observed for the group average of lymphocyte counts. On the other hand a statistically
significant decrease was observed in the group average of monocyte counts during the
treatment in all three groups. In men the count decreased after the first and second week of
treatment, while the change was not significant after the third week compared to the 0
point. At the individual level the monocyte count does not change or a slight decrease can
be observed. In men the group average of neutrophil granulocyte count increases after the
second and third week of treatment. At the individual level generally an increase can be
detected, but in a few cases a reduction was observed during the three weeks of the study.
The eosinophil count decreased for the whole group by the second and third week; in the
case of men the reduction was present already after the first week. There was no significant
change in the group average for women. Individually no change could be observed above
the uncertainty of the measurement.

The percentage of white blood cells shows a similar change to that of the absolute numbers.
The percent of monocytes decreased at the group level for all three groups already after the
first week of treatment. Further change was not observed. The percent of neurophils
increased for the whole group and for men after the second week of treatment, the percent
of eosinophils decreased in the whole group and in men already after the first week of the
treatment.

At the group level there were no changes in the red blood cell count and hemoglobin
content. After the second week of the treatment a slight decrease in hemocrit was observed
for the whole group. The average volume of erythrocytes (MCV) showed a very slight
decrease by the second week, therefore the hemoglobin concentration for one erythrocyte
(MCH) and the average hemoglobin concentration of the erythrocytes (MCHC) increased to a
small extent.

A statistically significant increase in the group average of thrombocyte count was observed
after two and three weeks of treatment both for the whole group and in men. Examining the
individual results, the subjects usually did not show large changes in the thrombocyte count,
and the thrombocyte count always remained within the normal range.

Biologically significant change was not observed in the qualitative and quantitative blood
count either at group or individual level.
2) Determination of immune parameters

The measurements carried out with the flow cytometer produced very similar results to
those carried out with the automated analyser regarding the percentile distribution of
lymphocytes, monocytes, neutrophil- and eosinophil granulocytes. This can be considered as
the internal control of the measurements.

The group averages of immune parameters are shown in table 3, the individual results in
table 4. The percentage of monocytes decreases at group level for the whole group and for
men by the second week of the treatment. At the same time the percentage of neutrophil
granulocytes increases at group level for the whole group and for men by the second week
of the treatment. The percent of eosinophils decreases in the whole group from the first
week of the treatment, and in the case of men by the second week of the treatment the
decrease is significant. In women the above parameters do not change significantly. In the
course of the treatment the ratio of leukocytes changes statistically, which could be
indicative, but the changes are so small that probably no physiological importance can be
attached to them.

No significant changes were observed in the percentage of total T cells, helper T cells,
immature T cells and B lymphocytes. The ratio of helper and cytotoxic cells did not change
(Th/Tc) either. In the case of men the ratio of cytotoxic T cells showed a small, but significant
reduction after the third week of the treatment. The percentage of NK-cells increased
significantly after the second week of the treatment both for the whole group and for
women. In men an increase was observed, but due to the large deviations in individual
results, the change was not significant statistically. Individually, in general either there was
no change or an increase was observed during the three weeks of the study. Although the
ratio of cytotoxic T lymphocytes showed a significant decrease, at the individual level the
changes were so small, that a physiological effect cannot be expected. Relatively bigger
changes (increase) were observed in the ratio of NK-cells at the individual level, compared to
the 0 point, which may have a functional impact: more NK cells are available to kill virally
infected or cancerous cells.

The percentage of activated (CD25+) T lymphocytes increased by the second and third week
in the whole group and in men. At the individual level there is either no change or an
increase can be observed, but in a few cases the percentage of activated T cells decreased in
the three weeks of the study. The percentage of activated (CD25+) helper T cells increased
for the whole group by the second week of the treatment. In general individually there is
either no change or an increase can be observed. The percentage of activated (CD25+)
cytotoxic T cells increased significantly after the third week in the case of men. The increase
in the expression of the CD25 cell surface molecule indicates the activation of T
lymphocytes. These results indicate the intensification of the cellular immune response.

The percentage of transferrin receptor positive (CD71+) T lymphocytes did not change
during the treatment. The percentage of B lymphocytes expressing transferrin receptors
(CD71+) decreased significantly by the second week in the whole group, and by the third
week this value returned to the original level. The individual data show such a large
distribution both individually and intra-individually that a biologically relevant conclusion
cannot be drawn from these data.

Among the examined persons, there was a man whose percentage of B lymphocytes was
well below the reference value. The reference range for B lymphocytes is 7.0-23%. The B cell
percentage of the person indicated as Q3,Q33,Q63,Q93 (Gábor Rabb) was between 0.3-0.9%
during the period of the study. The white blood cell count and the absolute lymphocyte
count did not decrease, but the percentage lymphocytes was low measured with both test
methods, and the percent of B lymphocytes was extremely low. The B cell count (data
calculated with the aid of the absolute number of lymphocytes and the percentage of B cells)
was at least one order of magnitude less than in the case of the other subjects. His data
were not included in the statistical analysis of immune parameters, as in our opinion they
would have falsified the data.
On the 15th of June 2009 the blood sample of the person coded Q35 (István Berei) deviated
to such an extent from the values measured during the three other occasions regarding
certain parameters (percentage of lymphocytes measured both with the automated
instrument and flow cytometer, percentage of helper T and NK-cells) that his data measured
on 15.06.2009 were omitted from the group level evaluation of the immune parameters.

3) Killing capacity of neutrophil granulocytes (production of reactive oxygen intermediates-

oxidative burst)

The group averages for the production of reactive oxygen intermediates of neutrophil
granulocytes are shown in table 5, the individual results in table 6. The reactive oxygen
intermediate production (ROI) of neutrophil granulocytes increased significantly in all three
groups from the first week of the treatment in the fMLP and PMA stimulated samples, and
from the second week in the samples stimulated with E. coli. Individually, in general an
increase was observed in ROI production, though in the samples stimulated with E. coli and
PMA a decrease relative to the 0 point was observed for certain individuals after the first
week.

The percentage of ROI producing cells increased significantly in all three groups from the
first week and this is also true at the individual level.

The increase in ROI production, and the fact that more cells respond to stimulation, result in
the increased killing potential (bactericidal effect) of neurophil granulocytes.
 Summary

1. No biologically significant changes were observed in the qualitative and quantitative blood
count either at group level or individual level during the 21 days of Kaqun treatment.

2. The percentage of NK-cells showed a statistically significant increase, and the individual
changes (increase) relative to the 0 point were bigger, which may have a functional
impact, namely that more NK cells are available to kill virus infected and cancerous cells.

3. A non-specific activation of T lymphocytes (indicated by the increase in the expression of

the CD25 cell surface antigen) could be detected, presumably caused by the Kaqun
treatment, indicating the increased activity of the cellular immune response.

4. Characteristically the value of several parameters changed significantly by the second

week of treatment and during the third week the value of the parameter remained at the
same level, or the change levelled to its original value (percent of neurophils, monocytes,
activated (CD25+) T cells, activated (CD25+) helper T cells and CD71+ B cells). This
suggests that two weeks treatment is the most effective for the change in immune
parameters and after that the reaction of the body to the treatment decreases, that is,
the effect cannot be boosted.

5. The increase of the production of reactive oxygen intermediates both at group level and
at the level of the individuals results in the intensification of the killing potential of
neurophil granulocytes.

28th of September 2009

Dr. Anna Biró

Head of department