biovis’D I A G N O S T I K

Nitrosative stress and/or mitochondrial co-factor deficiency

www.biovis.de

Expert information 1 /2012 Mitochondrial diseases and nitrosative stress

Mitochondrial DiseasesDiagnostics of acquired mitochondrial diseases and nitrosative stress

In the scope of mitochondrial diagnostics more and more parameters are offe-

red. Often they are expensive and elaborate procedures. But are those parame-

ters really better than those already available or do they simply provoke needless

expenses. biovis has revised its diagnostic spectrum in the range of mitochon-

drial medicine: sensible new parameters were included in our spectrum and old

pre-analytically very elaborate parameters were discontinued. This brochure is

the attempt to provide a guideline for mitochondria diagnostics. What is sensib-

le and what is not? Also pre-analytic snares are shown to avoid mistakes during

specimen collection, which may cause apparently pathological results.

Traditionally mitochondrial diseases are regarded as congenital diseases and

normally diagnoses are confirmed by muscle biopsy. There is a whole variety of

such inborn mitochondrial diseases. They can practically concern every area of

the mitochondrion. For pyruvate dehydrogenase alone there are several diseases

of which most are passed on dominantly x-chromosomal. In addition there are

also disorders of citrate cycle, the respiratory chain or fat burning (e.g. carnitin

transporter deficiency).

Acquired forms are more frequent than genetic mitochondrial diseases. For pa-

tients suffering from acquired mitochondrial diseases the analyses do not yield

characteristic findings. The clinical patterns may vary considerably. Patients of-

ten complain about lack of energy, but also complaints similar to those of CFS,

MCS or fibromyalgia can be observed.

Acquired mitochondrial diseases are triggered by nitrosative stress, which may

damage the structure and genome of mitochondria, and lack of mitochondrial

co-factors like for example co-enzyme Q10, riboflavine or niacin.

Mitochondrial Disease

In the scope of mitochondrial diagnostics more and more parameters are offe-

red. Often they are expensive and elaborate procedures. But are those parame-

ters really better than those already available or do they simply provoke needless

expenses. biovis has revised its diagnostic spectrum in the range of mitochon-

drial medicine: sensible new parameters were included in our spectrum and old

pre-analytically very elaborate parameters were discontinued. This brochure is

the attempt to provide a guideline for mitochondria diagnostics. What is sensib-

le and what is not? Also pre-analytic snares are shown to avoid mistakes during

specimen collection, which may cause apparently pathological results.

Traditionally mitochondrial diseases are regarded as congenital diseases and

normally diagnoses are confirmed by muscle biopsy. There is a whole variety of

such inborn mitochondrial diseases. They can practically concern every area of

the mitochondrion. For pyruvate dehydrogenase alone there are several diseases

of which most are passed on dominantly x-chromosomal. In addition there are

also disorders of citrate cycle, the respiratory chain or fat burning (e.g. carnitin

transporter deficiency).

Acquired forms are more frequent than genetic mitochondrial diseases. For pa-

tients suffering from acquired mitochondrial diseases the analyses do not yield

characteristic findings. The clinical patterns may vary considerably. Patients of-

ten complain about lack of energy, but also complaints similar to those of CFS,

MCS or fibromyalgia can be observed.

Acquired mitochondrial diseases are triggered by nitrosative stress, which may

damage the structure and genome of mitochondria, and lack of mitochondrial

co-factors like for example co-enzyme Q10, riboflavine or niacin.

Mitochondrial Disease

4 5

A whole variety of diseases or complaint patterns were associated with nitrosative

stress in the past. Inflammatory diseases promote the activation of inducible NO-

synthase (iNOS) by releasing cytokines. As a result more NO is developed – leading

to nitrosative stress.

A list of possible mitochondrial and chronic inflammatory diseases:

• Rheumatoidarthritis

• SeronegativeSpondyloarthropathy(e.g. Bekhterev’s disease, reactive arthritis)

• Connectivetissuediseases (e.g. systemic lupus, Sjögren’s syndrome, sclerodermia)

• Polymyalgiarheumatica

• Fibromyalgia

• Chronifiedinfections(e.g. borreliosis, chlamydia, hepatitis)

• Chronicfatigue

• Multiplesclerosis

• Tumourdiseases

• Metabolicsyndrome,diabetes,coronaryheartdiseaseand

allarteriosclerosisdiseases

• Secondarydepressionandanxiety

• Psoriasis

• Allergies,neurodermatitis,asthma

• Variousliverdiseases

• Migraine,chronicheadaches

• Chronicinflammatorygastro-intestinaldiseasesandirritablecolon

Diagnostics – Summary

Diagnostic approaches in case of mitochondrial diseases or nitrosative stress may

be manifold. They can be analyses of NO-development, preconditions of peroxi-

nitrite synthesis or impact on mitochondria. Also important therapeutic factors

have to be considered.

DeterminationofIncreasedNODevelopment • Citrulline in urine

• Amino acids in plasma (arginine and citrulline)

• NO – breath gas test

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

Nitrosative stress is generated if increased amounts of nitrogen monoxide (NO)

are developed. In its form as endothelial NO (eNOS) it leads to vascular vasodila-

tation. NO is developed in nerve cells (nNOS) in case of bacterial or viral infections

(iNOS) or in mitochondria (mNOS). NO is a colourless gas of radical nature because

of an unpaired electron. This is the cause of significant biological effects. In case

of oxidative stress it is converted to peroxynitrite after reactions with superoxide

anions. Peroxinitrite is a toxic substance which may damage mitochondria. It is the

main damaging agent in the cascade of nitrosative stress, mitochondrial disease,

immune dysfunction (chronic, often subclinical inflammation) and in many cases

also pains.

Frequent causes of increased NO synthesis or nitrosative stress are listed below.

Causes of nitrosative stress: [Pall et al. 2007, Kuklinski 2006, 2007]

• Pains

• ChronicInflammation

• chronicStress

• EnvironmentalToxins:solvents, pesticides, heavy metals

• Drugs: long-term nitrate, antihypertensive agents (e.g. dihydralazine),

cholesterol synthesis inhibitors, anti-diabetics (mainly metformin),

mitochondria damaging antibiotic agents (e.g. gentamicin, cotrimoxazole)

and others

• Cervical spine traumata

Nitrosative Stress

Complex I

H+

NADH + H+ NAD+

Succinate Fumarate

Pi + ADP ATP + H2O

1/2Os + H+ H2O

Complex II Complex III

H+

Complex IV

H+

ATP-Synthase

Biochemical consequences of increased NO syntheses

Inhibition of the FeS-containing enzymes in the mitochondrial respira-tory chain in complexes I and II reduced ATP development

H+

e- e-

e-

4 5

A whole variety of diseases or complaint patterns were associated with nitrosative

stress in the past. Inflammatory diseases promote the activation of inducible NO-

synthase (iNOS) by releasing cytokines. As a result more NO is developed – leading

to nitrosative stress.

A list of possible mitochondrial and chronic inflammatory diseases:

• Rheumatoidarthritis

• SeronegativeSpondyloarthropathy(e.g. Bekhterev’s disease, reactive arthritis)

• Connectivetissuediseases (e.g. systemic lupus, Sjögren’s syndrome, sclerodermia)

• Polymyalgiarheumatica

• Fibromyalgia

• Chronifiedinfections(e.g. borreliosis, chlamydia, hepatitis)

• Chronicfatigue

• Multiplesclerosis

• Tumourdiseases

• Metabolicsyndrome,diabetes,coronaryheartdiseaseand

allarteriosclerosisdiseases

• Secondarydepressionandanxiety

• Psoriasis

• Allergies,neurodermatitis,asthma

• Variousliverdiseases

• Migraine,chronicheadaches

• Chronicinflammatorygastro-intestinaldiseasesandirritablecolon

Diagnostics – Summary

Diagnostic approaches in case of mitochondrial diseases or nitrosative stress may

be manifold. They can be analyses of NO-development, preconditions of peroxi-

nitrite synthesis or impact on mitochondria. Also important therapeutic factors

have to be considered.

DeterminationofIncreasedNODevelopment • Citrulline in urine

• Amino acids in plasma (arginine and citrulline)

• NO – breath gas test

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

Nitrosative stress is generated if increased amounts of nitrogen monoxide (NO)

are developed. In its form as endothelial NO (eNOS) it leads to vascular vasodila-

tation. NO is developed in nerve cells (nNOS) in case of bacterial or viral infections

(iNOS) or in mitochondria (mNOS). NO is a colourless gas of radical nature because

of an unpaired electron. This is the cause of significant biological effects. In case

of oxidative stress it is converted to peroxynitrite after reactions with superoxide

anions. Peroxinitrite is a toxic substance which may damage mitochondria. It is the

main damaging agent in the cascade of nitrosative stress, mitochondrial disease,

immune dysfunction (chronic, often subclinical inflammation) and in many cases

also pains.

Frequent causes of increased NO synthesis or nitrosative stress are listed below.

Causes of nitrosative stress: [Pall et al. 2007, Kuklinski 2006, 2007]

• Pains

• ChronicInflammation

• chronicStress

• EnvironmentalToxins:solvents, pesticides, heavy metals

• Drugs: long-term nitrate, antihypertensive agents (e.g. dihydralazine),

cholesterol synthesis inhibitors, anti-diabetics (mainly metformin),

mitochondria damaging antibiotic agents (e.g. gentamicin, cotrimoxazole)

and others

• Cervical spine traumata

Nitrosative Stress

Complex I

H+

NADH + H+ NAD+

Succinate Fumarate

Pi + ADP ATP + H2O

1/2Os + H+ H2O

Complex II Complex III

H+

Complex IV

H+

ATP-Synthase

Biochemical consequences of increased NO syntheses

Inhibition of the FeS-containing enzymes in the mitochondrial respira-tory chain in complexes I and II reduced ATP development

H+

e- e-

e-

6 7

Special Diagnostics of Nitrosative Stress and Mitochondrial Diseases

Citrulline

Material: 1st morning urine

Standard: < 4 mg/g creatinine

Citrulline values above the standard indicate increased NO-accrual.

NO may lead to peroxynitrite development by reaction with super-

oxide anions. This might damage the mitochondria.

As citrulline development varies considerably (stress-dependent),

negative results do not rule out NO-stress or mitochondrial diseases.

The amount of citrulline developed depends on many omitting factors,

among others on the amount of available arginine.

Determinationofoxidativeandnitrosativestress • Lipid peroxidation, anti-oxidative capacity, glutathione

• Nitrotyrosine, nitrophenyl acetic acid

Determinationofmitochondrialdisorders • LDH isoenzyme

• Mitochondrial membrane potential

• Lactate Stress Test

• (Lactate / Pyruvate Ratio)

• M2PK (CAVE: is also a tumour marker)

DeterminationofInflammationandTH-Shift

• Humoral activity marker: CRP, neopterin, sIL2R, ECP

• Cytokines in serum, TNF, IL-1, IL-6 possible also IL-8 and IL-12

• Stimulated cytokine statuses

MicronutrientDiagnostics • Vitamin (mainly B12, B2, B3, folic acid, biotin , C, 25-OH-D3))

• Whole blood mineral analysis (mainly K, Mg, Zn, Se, amino acid status)

DeterminationofConsequences • Differential blood count, gGT, GPT, creatinine, urea, serum electrolytes,

blood lipids, blood sugar etc.

• L-tryptophan (alternatively serotonin), possibly complete aminogram

DeterminationofBlood-Brain-BarrierDisorders •Protein S100

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

Endogenic NO is developed by

the enzyme NOS (NO-Synthase)

from L-arginine.

Arginine + oxygen

NO + citrulline3-Nitrotyrosine

O2 Thiols

S-GSNOS-Nitrosothiol

ONOOPeroxinitrite

Superoxide anions

NO2Nitrite

NO

pH reduced

HN

CH2

CH2

CH2

H2N

OH

C

NH

CHC O

NH2

O

CH2

CH2

CH2

H2N

OH

C

NH

CHC O

NH2

NADPHNOS

l-Arginin

L-CitrullinNADP+ NADPH

H2O

NO-synthesis

FADFMN H4B

L-NMMAL-NAME

Pimagedine (aminoguanidine)

O2

6 7

Special Diagnostics of Nitrosative Stress and Mitochondrial Diseases

Citrulline

Material: 1st morning urine

Standard: < 4 mg/g creatinine

Citrulline values above the standard indicate increased NO-accrual.

NO may lead to peroxynitrite development by reaction with super-

oxide anions. This might damage the mitochondria.

As citrulline development varies considerably (stress-dependent),

negative results do not rule out NO-stress or mitochondrial diseases.

The amount of citrulline developed depends on many omitting factors,

among others on the amount of available arginine.

Determinationofoxidativeandnitrosativestress • Lipid peroxidation, anti-oxidative capacity, glutathione

• Nitrotyrosine, nitrophenyl acetic acid

Determinationofmitochondrialdisorders • LDH isoenzyme

• Mitochondrial membrane potential

• Lactate Stress Test

• (Lactate / Pyruvate Ratio)

• M2PK (CAVE: is also a tumour marker)

DeterminationofInflammationandTH-Shift

• Humoral activity marker: CRP, neopterin, sIL2R, ECP

• Cytokines in serum, TNF, IL-1, IL-6 possible also IL-8 and IL-12

• Stimulated cytokine statuses

MicronutrientDiagnostics • Vitamin (mainly B12, B2, B3, folic acid, biotin , C, 25-OH-D3))

• Whole blood mineral analysis (mainly K, Mg, Zn, Se, amino acid status)

DeterminationofConsequences • Differential blood count, gGT, GPT, creatinine, urea, serum electrolytes,

blood lipids, blood sugar etc.

• L-tryptophan (alternatively serotonin), possibly complete aminogram

DeterminationofBlood-Brain-BarrierDisorders •Protein S100

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

Endogenic NO is developed by

the enzyme NOS (NO-Synthase)

from L-arginine.

Arginine + oxygen

NO + citrulline3-Nitrotyrosine

O2 Thiols

S-GSNOS-Nitrosothiol

ONOOPeroxinitrite

Superoxide anions

NO2Nitrite

NO

pH reduced

HN

CH2

CH2

CH2

H2N

OH

C

NH

CHC O

NH2

O

CH2

CH2

CH2

H2N

OH

C

NH

CHC O

NH2

NADPHNOS

l-Arginin

L-CitrullinNADP+ NADPH

H2O

NO-synthesis

FADFMN H4B

L-NMMAL-NAME

Pimagedine (aminoguanidine)

O2

8

Compared to the LDH isoenzymes the analysis of the mitochondral

activity achieves results which can be quantified better – not only for

patient examinations, they can also be used also for in vivo or in vitro

studies (e.g. after giving mitotropic compounds like coenzyme Q10).

Attention: Measuring the mitochondrial activity has proved its

value over the last years. As no granulocytes are measured

there might be “paradox” results for tumour patients in rare cases –

when tumour patients with damaged specific immune defence and

prevailingly unspecific defence show apparently good values, although

apart from granulocytes the she situation looks different. In this case

ATP measurements are to be preferred.

LDH-Isoenzymes

Material: Serum, not frozen! (freezing destroys LDH-5)

Standard: LDH4: < 9,4 %

LDH5: < 10 %

LDH isoenzymes are reasonably priced markers for the evaluation of

mitochondrial functions. LDH-4 (H3M) and before all LDH-5 (4M) increase

if mitochondria are destroyed. Relative shares of LDH-4 or LDH-5

higher 10% indicate mitochondrial diseases (especially in case of normal

total LDH).

Attention: The total LDH should be within or only slightly above the standard range.

Severely increased total LDH may lead to misinterpretations, if liver

enzymes (gGT, GPT) and (heart) muscle enzymes (e.g. CK, troponin) as

well as haemolysis values (e.g. haptoglobin) are not available at the

same time, as LDH-5 increases are also found in case of liver damages.

There is also a relative shift to LDH1/2 in case of are myocardial damages

or haemolysis.

ATP-Measurement

Material: 1 CPDA (by express shipment because of living cell analysis)

Standard: > 500 pmol/ 106 cells

ATP-measurements are to be evaluated similar to those of the mito-

chondrial activity as ATP is developed in complex V of the respiratory

chain. Easily available leukocytes of blood are used for measuring

Nitrotyrosine,NitrophenylAceticAcid

Nitrotyrosine:

Material: EDTA blood (requires EXPRESS SHIPMENT)

Standard: <3.2 nmol/l

Peroxinitrite is toxic and may damage mitochondria. It cannot be

analysed directly. Peroxynitrite leads to nitrosation of aromatic amino

acids. For example 3-nitrotyrosine is developed from tyrosine, which

can be determined in blood. High nitrotyrosine levels indicate increased

peroxinitrite development and thus mitochondrial damage.

Increased nitrotyrosine values confirm the presence of nitrosative

stress (high specificity)

Attention: Just like in case of citrulline inconspicuous nitrotyrosine values do

not exclude mitochondrial damage by nitrosative stress. Reason for

inconspicuous measuring values may for example be tyrosine deficiency

Nitrophenylessigsäure:

Material: 1st morning urine

Standard: < 3,0 µg/g creatinine

3-nitrophenyl acetic acid is a metabolic product of 3-nitrotyrosine.

Positive nitrophenyl acetic acid values reliably confirm nitrosative

stress! Therefore measuring nitrophenyl acetic acid in urine is ana

logue to the nitrotyrosine determination in EDTA blood, but it is less

sensitive.

MitochondrialActivity

Material: EDTA blood (requires EXPRESS SHIPMENT)

Standard: > 90 % active mitochondria

Optimal: > 95 % active mitochondria

The enzymes of the respiratory chain transport protons from the cell

to the intermembrane space. This leads to development of electro-

chemical membrane potential, which can be determined with

fluorescence. Intact mitochondria show significant protone gradients,

inactive mitochondria do not. Flow cytometry procedures can determi

ne the membrane potential. Active and inactive mitochondria can

thus be distinguished. The mitochondrial activity is determined by mar

king granulocytes with a fluorescence colorant, which changes its

fluorescence depending on the electrical potential.

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

8 9

100

100

100 100 100 100

100

100

100

Green fluorescence (FL1)

Red

fluor

esce

nce

(FL2

) Staurosporine

Membrane potential can bemeasured by flow cytometry.

Cells with intact membrane potential

Cells with intact membra-ne potential

Increase of green florescence caused by mitochondrial function disorders

100

8

Compared to the LDH isoenzymes the analysis of the mitochondral

activity achieves results which can be quantified better – not only for

patient examinations, they can also be used also for in vivo or in vitro

studies (e.g. after giving mitotropic compounds like coenzyme Q10).

Attention: Measuring the mitochondrial activity has proved its

value over the last years. As no granulocytes are measured

there might be “paradox” results for tumour patients in rare cases –

when tumour patients with damaged specific immune defence and

prevailingly unspecific defence show apparently good values, although

apart from granulocytes the she situation looks different. In this case

ATP measurements are to be preferred.

LDH-Isoenzymes

Material: Serum, not frozen! (freezing destroys LDH-5)

Standard: LDH4: < 9,4 %

LDH5: < 10 %

LDH isoenzymes are reasonably priced markers for the evaluation of

mitochondrial functions. LDH-4 (H3M) and before all LDH-5 (4M) increase

if mitochondria are destroyed. Relative shares of LDH-4 or LDH-5

higher 10% indicate mitochondrial diseases (especially in case of normal

total LDH).

Attention: The total LDH should be within or only slightly above the standard range.

Severely increased total LDH may lead to misinterpretations, if liver

enzymes (gGT, GPT) and (heart) muscle enzymes (e.g. CK, troponin) as

well as haemolysis values (e.g. haptoglobin) are not available at the

same time, as LDH-5 increases are also found in case of liver damages.

There is also a relative shift to LDH1/2 in case of are myocardial damages

or haemolysis.

ATP-Measurement

Material: 1 CPDA (by express shipment because of living cell analysis)

Standard: > 500 pmol/ 106 cells

ATP-measurements are to be evaluated similar to those of the mito-

chondrial activity as ATP is developed in complex V of the respiratory

chain. Easily available leukocytes of blood are used for measuring

Nitrotyrosine,NitrophenylAceticAcid

Nitrotyrosine:

Material: EDTA blood (requires EXPRESS SHIPMENT)

Standard: <3.2 nmol/l

Peroxinitrite is toxic and may damage mitochondria. It cannot be

analysed directly. Peroxynitrite leads to nitrosation of aromatic amino

acids. For example 3-nitrotyrosine is developed from tyrosine, which

can be determined in blood. High nitrotyrosine levels indicate increased

peroxinitrite development and thus mitochondrial damage.

Increased nitrotyrosine values confirm the presence of nitrosative

stress (high specificity)

Attention: Just like in case of citrulline inconspicuous nitrotyrosine values do

not exclude mitochondrial damage by nitrosative stress. Reason for

inconspicuous measuring values may for example be tyrosine deficiency

Nitrophenylessigsäure:

Material: 1st morning urine

Standard: < 3,0 µg/g creatinine

3-nitrophenyl acetic acid is a metabolic product of 3-nitrotyrosine.

Positive nitrophenyl acetic acid values reliably confirm nitrosative

stress! Therefore measuring nitrophenyl acetic acid in urine is ana

logue to the nitrotyrosine determination in EDTA blood, but it is less

sensitive.

MitochondrialActivity

Material: EDTA blood (requires EXPRESS SHIPMENT)

Standard: > 90 % active mitochondria

Optimal: > 95 % active mitochondria

The enzymes of the respiratory chain transport protons from the cell

to the intermembrane space. This leads to development of electro-

chemical membrane potential, which can be determined with

fluorescence. Intact mitochondria show significant protone gradients,

inactive mitochondria do not. Flow cytometry procedures can determi

ne the membrane potential. Active and inactive mitochondria can

thus be distinguished. The mitochondrial activity is determined by mar

king granulocytes with a fluorescence colorant, which changes its

fluorescence depending on the electrical potential.

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

8 9

100

100

100 100 100 100

100

100

100

Green fluorescence (FL1)

Red

fluor

esce

nce

(FL2

) Staurosporine

Membrane potential can bemeasured by flow cytometry.

Cells with intact membrane potential

Cells with intact membra-ne potential

Increase of green florescence caused by mitochondrial function disorders

100

cellular ATP. Apparently paradoxical results in case of tumour patients –

as can be observed in mitochondrial activity measurements – do not

occur when measuring cellular ATP.

Basal ATP (initial measurement) is measured first. Then sodium azide

is added to the cells. This reversibly inhibits the synthesis chain in

complex IV. Due to the inhibition the ATP concentration considerably

decreases (Stress ATP). If the inhibitor is now removed again, the elec-

tron transport chain starts to regenerate and ATP development in-

creases (Recovery-ATP). While patients frequently show normal basal

ATP values, the mitochondrial regeneration capacity is significantly

limited in many cases. The recovery-ATP should be at least 25 %

above that of the stress-ATP value. Increases of 50 % and more can

often be observed in healthy people. If increases of 25% are not reached,

one can safely assume disordered mitochondrial regeneration.

ATP-measurementsprovideinformationaboutmitochondrialfunction,

capacityandperformance.

Lactate / Pyruvate Ratio

Material: 1-2 NaF (ship via express)

Attention: As food consumption and physical exercise lead to significant increase

of pyruvate values, the sample has to be taken on an empty

stomach and in resting condition. Stasis should not be longer than

1 minute.

Standard: Ratio < 20:1

Thepyruvatedeterminationisextremelysusceptibletofailure(food,

exercise,bloodstasisinveins)andalsoproblematicwheretransportis

concerned.Duetotheinstabilityofthesamplematerialthismitochon-

drialfunctiontestisonly recommended with restrictions.

ProteinS100

Material: 2 x serum

• blood withdrawal in resting condition

• 10 minutes climbing stairs or head circling

• Renewed blood withdrawal

• Centrifuge whole blood and freeze serum, ship in frozen condition

per express

Standard: > 0.13 (malign melanoma) / > 0.07 µg/l (nitrosative stress)

Each value higher than 0.07 µh/l indicates blood-brain barrier

disorders.

In case of moderate complaints protein S 100 will normally only increase

after strain (climbing stairs, circling head).

Causes of non-specific increases of protein S100 – values

• slight increases: liver cirrhosis, renal insufficiency

• increases up to 2.0 µg/l: severe bacterial infections.

In case of physiologically dark skin colour: Protein S 100 test cannot

be evaluated!!!

• Significant increase (more than 2.0 µg/l): vascular damage,

heart attack, cerebral ischaemia

Attention: Haemolysis leads to false positive results.

OrganicAcidsinUrine

Material: Urine

Profile: Organic Acids of the Citric Acid Cycle

One of the major consequences of nitrosative stress is the destruction

of iron containing enzymes. One of these enzymes is aconitase, which

catalyses the conversion of citric acid (citrate) to isocitric acid (isocitrate).

If the aconitase is destroyed by peroxynitrite this will lead to

citric acid stasis and an interruption of the cancer cycle. Increased

citric acid and simultaneously low isocitric acid may therefore be an

additional indication of mitochondrial diseases.

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

10 11

Patient 1

(ATP

)

435pmol

NW: 500 - 1100 Pmol

980pmol

658pmol

Patient 3Patient 2

(ATP

)

1050pmol

40,4 %

100 %

84,5 %

424pmol

887pmol

+ 109 %

Initial Measurement Stress Measurement Regeneration Measurement

Initial Measurement Stress Measurement Regeneration Measurement

(ATP

)950

pmol

35,9 %

100 %

40,5

341pmol

385pmol

+ 12,8 %

Mitochondria – ATP – Initial Measurement

Mitochondria – Stress Test - satisfactory

Mitochondria – Stress Test - deficient

cellular ATP. Apparently paradoxical results in case of tumour patients –

as can be observed in mitochondrial activity measurements – do not

occur when measuring cellular ATP.

Basal ATP (initial measurement) is measured first. Then sodium azide

is added to the cells. This reversibly inhibits the synthesis chain in

complex IV. Due to the inhibition the ATP concentration considerably

decreases (Stress ATP). If the inhibitor is now removed again, the elec-

tron transport chain starts to regenerate and ATP development in-

creases (Recovery-ATP). While patients frequently show normal basal

ATP values, the mitochondrial regeneration capacity is significantly

limited in many cases. The recovery-ATP should be at least 25 %

above that of the stress-ATP value. Increases of 50 % and more can

often be observed in healthy people. If increases of 25% are not reached,

one can safely assume disordered mitochondrial regeneration.

ATP-measurementsprovideinformationaboutmitochondrialfunction,

capacityandperformance.

Lactate / Pyruvate Ratio

Material: 1-2 NaF (ship via express)

Attention: As food consumption and physical exercise lead to significant increase

of pyruvate values, the sample has to be taken on an empty

stomach and in resting condition. Stasis should not be longer than

1 minute.

Standard: Ratio < 20:1

Thepyruvatedeterminationisextremelysusceptibletofailure(food,

exercise,bloodstasisinveins)andalsoproblematicwheretransportis

concerned.Duetotheinstabilityofthesamplematerialthismitochon-

drialfunctiontestisonly recommended with restrictions.

ProteinS100

Material: 2 x serum

• blood withdrawal in resting condition

• 10 minutes climbing stairs or head circling

• Renewed blood withdrawal

• Centrifuge whole blood and freeze serum, ship in frozen condition

per express

Standard: > 0.13 (malign melanoma) / > 0.07 µg/l (nitrosative stress)

Each value higher than 0.07 µh/l indicates blood-brain barrier

disorders.

In case of moderate complaints protein S 100 will normally only increase

after strain (climbing stairs, circling head).

Causes of non-specific increases of protein S100 – values

• slight increases: liver cirrhosis, renal insufficiency

• increases up to 2.0 µg/l: severe bacterial infections.

In case of physiologically dark skin colour: Protein S 100 test cannot

be evaluated!!!

• Significant increase (more than 2.0 µg/l): vascular damage,

heart attack, cerebral ischaemia

Attention: Haemolysis leads to false positive results.

OrganicAcidsinUrine

Material: Urine

Profile: Organic Acids of the Citric Acid Cycle

One of the major consequences of nitrosative stress is the destruction

of iron containing enzymes. One of these enzymes is aconitase, which

catalyses the conversion of citric acid (citrate) to isocitric acid (isocitrate).

If the aconitase is destroyed by peroxynitrite this will lead to

citric acid stasis and an interruption of the cancer cycle. Increased

citric acid and simultaneously low isocitric acid may therefore be an

additional indication of mitochondrial diseases.

biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss

10 11

Patient 1

(ATP

)

435pmol

NW: 500 - 1100 Pmol

980pmol

658pmol

Patient 3Patient 2

(ATP

)

1050pmol

40,4 %

100 %

84,5 %

424pmol

887pmol

+ 109 %

Initial Measurement Stress Measurement Regeneration Measurement

Initial Measurement Stress Measurement Regeneration Measurement

(ATP

)

950pmol

35,9 %

100 %

40,5

341pmol

385pmol

+ 12,8 %

Mitochondria – ATP – Initial Measurement

Mitochondria – Stress Test - satisfactory

Mitochondria – Stress Test - deficient

Diagnostik MVZ GmbH

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