2
• To be the referring point of the oxidative
stress measurement:
– Measurement of free radicals: d-ROMs test
– Measurement of antioxidant potential: BAP
test
– Interpretation of the results and oxidative
stress diagnosis: Win Os Manager
Our Mission
3
History of Oxidative Stress Measurement
• In the first 90s the researcher Mauro Carratelli invented
the d-ROMs test, which started to the “oxidative stress
era”. The finalization of the BAP test later followed.
• Fabrizio Callegari believed that to spread the test it was
necessary to carry out the test in real time in the (very)
medical office rooms.
4
• As a consequence FRAS –Free Radical Analytical System –came into existence to allow the measurement of free radicals from a drop of capillary blood.
• From the projects of Mauro Carratelli and FabrizioCallegari a new challenge was born and turned into reality: H&D srl, a team of professional people leader in the measurement of oxidative stress
5
Oxidative Stress
• In all living organisms there is a fragile balance between the production and the elimination of free radicals through the activity of the antioxidant systems.
• The loss of this balance, indicated as oxidative stress, causes the start of cellular lesions.
• If these cellular lesions are severe and continue in time, the outcome will be an acceleration of the natural aging process and the onset of many diseases.
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• Oxidative stress = Cellular damage
• Cellular damage = Tissue damage
• Tissue damage = Organ damage
• Organ damage = Disease
• Disease = Prescription drug
• Prescription drug = Damaging side effects
Oxidative Stress
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Oxidative Stress
• It is obvious that it is already too late when you intervene on the recognized pathology.
• You must even accept that to cure this pathology it is probable to have damaging side effects.
• The winning strategy is to intervene since the very beginning when the cellular damage originates, that is when oxidative stress starts to manifest and therefore it is measurable.
8
Oxidative Stress
• H&D makes it possible to intervene in this
preventive direction, making available to
doctors and health
operators the
FRAS 4
9
Oxidative Stress
• FRAS 4 allows to evaluate in a comprehensive way the presence of oxidative stress through the d-ROMs and the BAP test , which can be carried in real time in the rooms of the medical doctor and make possible a precise and reliable diagnosis of oxidative stress.
• The two overlapping components of oxidative stress, the pro and anti-oxidant components, will be evaluated singularly and the meaning of their possible combination carefully considered.
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Oxidative Stress
• Everybody should take the “Oxidative Stress
Global Evaluation” in healthy conditions or if
there is an exposition to pro-oxidation elements
(i.e. unhealthy life stile, environment pollution…)
or if there is some chronic pathology (i.e.
diabetes, atherosclerosis, dementia, rheumatic
arthritis…) or if there the impelling necessity to
have some treatments (i.e. dialysis, by-pass,
organ transplantation, contraceptive pill,
radiotherapy, chemotherapy…)
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Oxidative Stress
• It is possible only through the Oxidative Stress
Global Evaluation to optimize and monitor the
real efficacy of antioxidant formulations, which
too often are taken
by the individuals
without assessing
the real necessity
of it.
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FRAS 4 – Free Radical Analytical System
• FRAS 4 is the latest product by H&D and it is available to doctors and health operators allowing the Oxidative Stress Global Evaluation with the d-ROMs and BAP tests.
• FRAS 4 is a dedicated photometer with inbuilt centrifuge allowing the operator to carry out the d-ROMs and BAP test in a simple and self-guided way through the help of the messages appearing successively on the instrument display.
• FRAS 4 prints a ticket with the value and the date of the test. The ticket can be personalized with the name and the address of the doctor.
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FRAS 4 – Free Radical Analytical System
• Technical features
RS 232 with 9 poles for PC connectionInterface
Resident on FLASH memory (2)Program
Software
37°C real time displayedTemperature
2 – 4Housings
6000 r. p. m. ± 5%Speed
Centrifuge
37°C real time displayedReading cell
Absorbance. Lambert Beer law.Measuring principle
505 nm obtained with interferential filter(1) Spectral region
Long life focussed lend-end lampLamp
Photometric system
50 W Power consumption
100 ÷ 240 VAC, 50 ÷ 60 Hz Power supply
Approx 3,9 kg Weight
39 x 26 x 12 cm Dimension
General characteristics
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FRAS 4 – Free Radical Analytical System
• Technical features
Up to 90%Relative humidity
DIRECTIVE 73/23/CEESafety
(1) With 8 nm bandwidth
(2) Updatable through a serial PC port
(3) With 4 rows and 20 characters
(4) Customizable, with the reprint options
(5) Regulations: CEI-EN 61010-1, CLASS I, INSTAL. CATEG II
DIRECTIVE 89/336/CEEElectromag. Compat.
Room temperature, 15 – 35° CTemperature
Working conditions
Automatic error display
Autodiagnosis
Automatic printout(4)Results
Graphic, thermic, with 192 dots per lineType
Printer
Back lighted alphanumeric LCD(3)
Display
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d-ROMs Test• d-ROMs test is a reliable, repeatable and precise test which allows the
determination of the pro-oxidant status of a living being through the measurement of the haematic concentration of the ROS (Reactive Oxygen Metabolites = Free Radicals).
• Italian CNR (National Centre for Research) has confirmed the complete superimposition of the values of free radicals obtained with the d-ROMs test and with the “golden standard” (ESR - Electron Spin Resonance).
0 25 10050
0
1.0
0.5
Time (min)
EPR intensity(arbitrary units)
75 0 25 10050
0
0.8
0.4
Absorbanceat 505 nm(A
505)
75
Time (min)
(A) Room temperature time profile of the normalised spectral intensity (•) and of A505 readings (�) exhibited by the system DEPPD (3.7 x 10-3 M)/tBuOOH (3.9 x 10-5 M)/FeSO4 (2.8x10-5 M) at room temperature. (B) Time profile of the A505 readinmgs exhibited by the systems DEPPD (3.7 x 10-3
M)/tBuOOH (3.9 x 10-5 M)/FeSO4 (2.8x10-5 M) (•), DEPPD (3.7 x 10-3 M)/tBuOOH (2.0 x 10-5 M)/FeSO4
(2.8x10-5 M) (�) and DEPPD (3.7 x 10-3 M)/tBuOOH (0.95 x 10-5 M)/FeSO4 (2.8x10-5 M) (�) at room temperature. tBuOOH: tert-buthylhydroperoxide; DEPPD: N,N-diethylparaphenylendiamine
0 25 10050
0
1.0
0.5
Time (min)
EPR intensity(arbitrary units)
75 0 25 10050
0
0.8
0.4
Absorbanceat 505 nm(A
505)
75
Time (min)
(A) Room temperature time profile of the normalised spectral intensity (•) and of A505 readings (�) exhibited by the system DEPPD (3.7 x 10-3 M)/tBuOOH (3.9 x 10-5 M)/FeSO4 (2.8x10-5 M) at room temperature. (B) Time profile of the A505 readinmgs exhibited by the systems DEPPD (3.7 x 10-3
M)/tBuOOH (3.9 x 10-5 M)/FeSO4 (2.8x10-5 M) (•), DEPPD (3.7 x 10-3 M)/tBuOOH (2.0 x 10-5 M)/FeSO4
(2.8x10-5 M) (�) and DEPPD (3.7 x 10-3 M)/tBuOOH (0.95 x 10-5 M)/FeSO4 (2.8x10-5 M) (�) at room temperature. tBuOOH: tert-buthylhydroperoxide; DEPPD: N,N-diethylparaphenylendiamine
0 25 10050
0
1.0
0.5
Time (min)
EPR intensity(arbitrary units)
75 0 25 10050
0
0.8
0.4
Absorbanceat 505 nm(A
505)
75
Time (min)
0 25 10050
0
1.0
0.5
Time (min)
EPR intensity(arbitrary units)
75 0 25 10050
0
0.8
0.4
Absorbanceat 505 nm(A
505)
75
Time (min)
(A) Room temperature time profile of the normalised spectral intensity (•) and of A505 readings (�) exhibited by the system DEPPD (3.7 x 10-3 M)/tBuOOH (3.9 x 10-5 M)/FeSO4 (2.8x10-5 M) at room temperature. (B) Time profile of the A505 readinmgs exhibited by the systems DEPPD (3.7 x 10-3
M)/tBuOOH (3.9 x 10-5 M)/FeSO4 (2.8x10-5 M) (•), DEPPD (3.7 x 10-3 M)/tBuOOH (2.0 x 10-5 M)/FeSO4
(2.8x10-5 M) (�) and DEPPD (3.7 x 10-3 M)/tBuOOH (0.95 x 10-5 M)/FeSO4 (2.8x10-5 M) (�) at room temperature. tBuOOH: tert-buthylhydroperoxide; DEPPD: N,N-diethylparaphenylendiamine
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d-ROMs Test
• The results are expressed in Carratelli Units, in short U
Carr, which are the measuring units for free radicals,
adopted and recognized by the international scientific
community.
• The d-ROMs test is covered by international patents.
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d-ROMs Test
• The normality range for d-ROMs test was obtained by
examining 5.000 clinically healthy subjects and is
between 250 and 300 U Carr. This means that healthy
population has a value of d-ROMs, or normal level of
free radical activity, between 250 and 300 U Carr.
Middle level of oxidative stress341-400
Unit of measurement U. Carr
1 U. Carr = 0.08 mg H2O2/dl
Very high level of oxidative stress> 500
High level of oxidative stress401-500
Low level of oxidative stress321-340
Border condition300-320
Normal range250-300
d-ROMs test
REFERENCE VALUES
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d-ROMs Test
• To carry out the d-ROMs test it is necessary a little drop
of capillary blood (20 micro-litre) taken from the fingertip.
• The d-ROMs kit contains all the necessary items to carry
out 50 tests.
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BAP Test
Biological Antioxidant Potential
• The BAP test is a reliable, repeatable and precise test which
allows determining the haematic concentration of the anti-
oxidant compounds.
• The BAP test has been validated through the comparison with
the “golden standard” (ESR - Electron Spin Resonance) by
the Showa University School of Medicine in Tokyo.
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BAP Test
Biological Antioxidant Potential
• The BAP test is thus a test able to determine in a specific
manner the scavenging/antioxidant activities in living beings.
• The normality value for the BAP test was obtained by
examining a large population of healthy subjects and is more
than 2.200 micro-mol/litres. This means that healthy
population has a value of BAP above 2.200 micro-mol/litres.
µMol/L
Veri severe shortage< 1400
Severe shortage1600 – 1400
Shortage1800 – 1600
Moderate shortage2000 – 1800
Border line2200 – 2000
Optimum value2200 – 4000
BAP test
REFERENCE VALUES
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BAP Test
Biological Antioxidant Potential
• To carry out the BAP test you need 10 micro-litres of
plasma obtained by the centrifugation of one drop of
capillary blood taken from the fingertip.
• The BAP kit contains all the necessary items to carry out
50 tests.
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Win OS Manager
• The interpretation of the values of d-ROMs test and of BAP test is delegated to the doctor.
• The crossing of these two values (normal, low or high) must be interpreted with the help of the Guide Lines (available now to the doctor), prepared by Prof. Eugenio Luigi Iorio, President of the International Observatory of Oxidative Stress.
• This interpretation leads to the diagnosis of oxidative stress.
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Win OS Manager
• A specific software has been created to help the doctor in the diagnosis: Win Os Manager which allows to:– Save and file all the results of the tests
– Interpret the situation and the risk of oxidative stress
– Consider the whole clinical situation of that particular patient in relation to the results obtained
– Suggest eventual therapies with vitamins and antioxidants
– Highlight the date for the next oxidative stress check up
– Print out diversified reports for the doctor and for the patient.
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Win OS Manager
• The software presents some “utilities”
offering the doctor easy access to
important information such as:
– the specific diagnostic algorithm
– the ORAC point system of the most common
vegetables
– RDA tables
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Book of References
• The Oxidative Stress Global Evaluation with the d-ROMs and BAP tests has been adopted in the entire world by hundreds of researchers and is now available for the doctors and for the health operators.
• Every doctor and health operator can now intervene in a preventive manner on the onset of pathologies and monitor the intake of antioxidant supplements to reach the real well-being of the patient.
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Book of References
• Researchers produced a large number of studies by using the d-ROMs and BAP tests and published them all over the world. Over 580 worldwide studies are collected in our Book of Reference.
• In the Book of Reference studies are divided by year of publication, by alphabetical order of author and by medical specialization.
• The d-ROMs and BAP tests are the only tests recommended by the International Observatory of Oxidative Stress.
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