Brochure Oxidative Stress March 2019 · 3 Oxidative stress – underlying cause of many diseases Oxidative stress represents an imbalance between the systemic manifestation of reactive

Oxidative and antioxidative status

Radical generating and degrading enzymes

Antioxidants

Modified molecules (lipids, proteins, sugar moieties, DNA)

Genetic predispositions

Additional biomarkers for oxidative stress

Oxidative Stress

Test Kits for Research & Routine

Content ADMA /ADMA Xpress ...........................................................................................31AOPP ..........................................................................................................................23Carbonyl Protein .....................................................................................................22L-Citrulline ................................................................................................................34C-reactive Protein (CRP) ......................................................................................35Glutathione (GSH/GSSG)......................................................................................16Haptoglobin .............................................................................................................11ImAnOx® (TAS/TAC) ................................................................................................. 6Malondialdehyde (MDA) ......................................................................................21Myeloperoxidase (IDK® MPO)............................................................................... 8Nitric Oxide Synthase 3 (NOS3) .........................................................................29Nitrotyrosine ............................................................................................................248-OHdG ......................................................................................................................25Oxidiertes LDL (ox-LDL) / MDA Addukt ..........................................................18ox-LDL Autoantibodies (anti ox-LDL) ..............................................................20PerOx (TOS/TOC) ...................................................................................................... 5PMN Elastase (IDK® PMN Elastase) ...................................................................36Relaxin ........................................................................................................................37SDMA ..........................................................................................................................33Thiol-Status ...............................................................................................................38Ubiquinone (Coenzyme Q10) .............................................................................15Vitamin A/E ...............................................................................................................14Vitamin C ...................................................................................................................12

Molecular biology tests (PCR)MutaCHIP® TOXO ....................................................................................................29MutaPLATE® GST-M1/T1 ......................................................................................30MutaPLATE® GST-P1 ...............................................................................................30MutaGEL® OxStress I ..............................................................................................27MutaGEL® OxStress II ........................................................................................9, 28

1. Oxidative and antioxidative status .........4

2. Radical generating and degrading enzymes ...........7

3. Antioxidants .......................10

4. Modified molecules (lipids, proteins, sugar moieties, DNA) ............. 17

5. Genetic predispositions ................ 26

6. Additional biomarkers of oxidative stress .................. 31

US: all products: Research Use Only. Not for use in diagnostic procedures.

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Oxidative stress – underlying cause of many diseases Oxidative stress represents an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Am imbalance in the normal redox state of cells can cause toxic eff ects through the production of per-oxides and free radicals leading to cell damage. The damage includes oxidation of proteins, lipids, and DNA thus altering, often irreversibly, key structures of cells. Many factors such as infl ammation, malnutrition, excessive sports, stress, drugs or environmental toxins can shift the oxidative balance unfavourably - with health threatening eff ect: Oxidative stress is considered a signifi cant factor for the develop-ment and manifestation of many diseases. These include:

1. Cancer 2. Neurological and psychiatric diseases such as Parkinson's and Alzheimer's

disease, schizophrenia, bipolar disorder, fragile X syndrome, autism, and chronic fatigue syndrome

3. Cardiovascular symptoms and diseases such as endothelial dysfunction, athero-sclerosis, hypertension, heart failure, myocardial infarction

4. Diabetes and diabetes related end-organ damage5. Chronic renal failure 6. Autoimmune diseases 7. Premature aging8. Skin diseases: Lichen planus, vitiligo

However, reactive oxygen species are also needed in a certain amount to stay healthy. They belong to the non-specifi c defense system of our immune system and are used as a way to attack and kill pathogens. Antioxidants like vitamines shield us from the negative impact of ROS by functioning as radical scavengers, thereby establishing and maintaining an oxidative balance. Taken together, the right balance between reactive oxygen species and antioxidants is critical for a healthy life.

Immundiagnostik off ers a broad portfolio for a comprehensive laboratory analysis of oxidative stress. Our tests enable monitoring of the oxidative stress exposure on various levels with diff erent parameters.

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1. Oxidative and antioxidative statusReactive oxygen species (ROS) are generated in living organisms by specifi c enzymes. The overall capacity to generate ROS is called oxidative status. The counterpart of this system is the antioxidative system which consists of enzyma-tic and non-enzymatic antioxidants. Both, the activity of antioxidative enzymes and the capacity of non-enzymatic antioxidants are known as the antioxidative status of the organism. If the balance between prooxidative processes and anti-oxidative system is disturbed oxidative stress occurs. To get fi rst overall insights wether oxidative stress may play a role in certain diseases, an analysis of the oxi-dative and anti-oxidative status is very helpful. In particular, the relationship of both overall oxidative capacity and overall anti-oxidative capacity may provide fi rst hints of clinically relevant oxidative stress.

Our photometric test systems provide an insight into the individual ratio of oxidative and antioxidative processes. In case of an imbalance, the underlying cause(s) can be determined subsequently by analyzing specifi c single parameters.

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1. Oxidative and antioxidative status

PerOx (TOS/TOC)(Oxidative Status)

The currently established methods for the measurement of effects induced by radicals, e.g. lipid peroxidation, are in part designed for assays performed on a large scale or are only suitable for the measurement of disgraduation products of polyun-saturated fatty acids, e.g. thiobarbituric acid reacting substances (TBARS).

Our PerOx(TOS, "total oxidative status") assay in contrast is fast and easy-to-use and could be used to measure the total lipidperoxides. Because of the direct correla-tion between free radicals and lipid peroxides, the whole oxidative status/oxidative stress of the sample can be determined and characterised.

Indications • Cardiovascular disease • Aging • Inflammatory processes • Carcinogeneses

ReferencesAlexopoulos N et al. (2008) Eur J Cardiovasc Prev Rehabil 15(3):300-5Li H et al. (2008) J Pharmacol Exp Ther 326(3):745-53Bader N et al. (2007) Br J Nutr 98(4):826-33Koenig B et al. (2007) Implants: 20-27Schönermarck U et al. (2006) Clin Nephrol 66(5):357-63 Schulpis KH et al. (2006) Toxicology 217(2-3):228-32 Lindschinger M et al. (2004) Clin Chem Lab Med 42(8):907-14Hildebrandt W et al. (2002) Blood 99:1552-5Schimke I et al. (2001) J Am Coll Cardiol 38:178-83.

Simple sample preparation

Short incubation times

Designed to be suitable for proces-sing small amounts of specimen

Determination of complete antioxidative capacity

Reference Values PerOx (TOS/TOC)

EDTA plasma / serum:

good < 200 µmol/llow oxidative stress 200 - 350 µmol/lhigh oxidative stress > 350 µmpl/l

PerOx (TOS/TOC)

Sample volume 20 µlMatrix Serum, EDTA plasmaTest principle colorimetricCat. No. KC 5100

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ImAnOx® (TAS/TAC)(Antioxidative capacity)

Overproduction of oxygen radicals or insufficient antioxidative defence mechanis-ms lead to a dangerous imbalance in the organism. This enduces several patho- mechanisms causing a large variety of diseases, e.g. cardiovascular disease and athe-rosclerosis. Additionally, they contribute to and/or cause, sepsis, malignancies, neuro-degenerative and inflammatory processes.

The antioxidative capacity assay ImAnOx® (TAS, "total antioxidative status") is fast and easy-to-use and is suitable for the meas

Indications

• Cardiovascular disease • Aging • Inflammatory processes • Carcinogeneses

ReferencesAvivi I et al. (2009) Bone Marrow Transplant 43(10):801-6Alexopoulos N et al. (2008) Eur J Cardiovasc Prev Rehabil 15(3):300-5Kiviniemi TO et al. (2008) Cardiovasc Ultrasound 6:25.Omurtag GZ et al. (2008) J Pineal Res 44(4):432-8Schulpis KH et al. (2008) Clin Chem Lab Med 46(5):680-6. Shahar E et al. (2008) Curr HIV Res 6(5):447-51Bader N et al. (2007) Br J Nutr 98(4):826-33Chrysohoou C et al. (2007) Atherosclerosis 192(1):169-76Kiviniemi TO et al. (2007) Atherosclerosis 195(2):176-81Koenig B et al. (2007) Implants: 20-27Hanimoglu H et al. (2007) Clin Neurol Neurosurg 109(7):561-6Hershkovich O et al. (2007) J Gerontol A Biol Sci Med Sci 62(4):361-6Panagiotakos DB et al. (2007) Diabetes Obes Metab 9(5):660-8Vlachopoulos C et al. (2007) Eur Heart J 28(17):2102-9.Schönermarck U et al. (2006) Clin Nephrol 66(5):357-63Schulpis KH et al. (2006) Toxicology 217(2-3):228-32 Stepan H et al. (2004) Ultrasound Obstet Gynecol 23(6):579-83


Short incubation times

Designed to be suitable for processing small amounts of

specimen

Determination of complete antioxidative capacity

Microtiterplate assay

Reference Values ImAnOx (TAS/TAC)

EDTA-Plasma / Serum:

low < 280 µmol/l acceptable 280 - 320 µmol/l good > 320 µmpl/l

ImAnOx® (TAS/TAC)

Sample volume 40 µlMatrix Serum, EDTA plasmaTest principle colorimetricCat. No. KC5200

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2. Radical generating and degrading enzymesThe balance between overall oxidative capacity and overall antioxidative capacity in living organisms is tightly controlled by enzymes that either catalyze the gene-ration of ROS or are involved in their degradation. If overall testing indicated that oxidative stress is present, it is often very helpful to get insights into the underlying processes by analyzing specific enzymes involded in the generation or degradation of ROS. This approach sometimes allows the detection of a specific defect either in the ROS generating enzyme system or in the ROS degradation system. The result might be helpful for the understanding of the pathogenesis of a certain disease and might also offer specific options for intervention.

Our ELISAs and colorimetric test systems determine the level of relevant enzymes in the circulation.

IDK® MPO (Myeloperoxidase)MPO is part of the defence mechanism of the polymorphonuclear leukocytes against exogenic substances. During bacterial infection, these leukocytes, stimulated by chemotactically effective substances (leukotrienes, complement factors, bacterial toxins etc.), move to the site of the infection where they encapsulate the foreign substances. If the foreign body is located in an intracellular vacuole, then various substances are used for the intracellular digestion. Amongst these are MPO, catio-nic proteins, lysozymes, lactoferrins and some acidic hydrolases. A strong surge of oxidative metabolism takes place, producing a high number of oxygen radicals that destroy the foreign proteins.

Some of these defence compounds leak into the extracellular space during this process. This happens to a greater extent when the leukocytes cannot en-capsulate the foreign body because of its size or in cases where the neutro-phils themselves are destroyed through bacterial toxins, crystalline substances etc.. MPO, together with H2O2 and a halogen, forms a very strong anti-microbial system, which can effectively combat a number of microorganisms.

MPO exists in high concentrations in neutrophilic leukocytes, whilst hydrogen peroxide production is only increased during the attacks or released by challenged microorganisms. Chloride, which is sufficiently available in leukocytes, is used as the required halogen. Iodine is about 100 times more effective, but is only present in serum in small concentrations. It can be replaced by hormones containing iodine, e.g. thyroxine and triiodothyronine. These are deiodised by leukocytes during pha-gocytosis.

The MPO system is inhibited by catalase and reducing agents (such as ascorbic acid or glutathione) that deactivate H2O2 its primary substrate. If any of these agents are missing, the MPO system can attack other cells in the extracellular space. These include spermatocytes, erythrocytes, leukocytes and tumor cells. The mechanism of the system involves halogenation of proteins and the production of highly reactive singulet oxygen. MPO is used by the neutrophil granulocytes to oxidize chloride ions via H2O2. The resulting hypochloride acid is also an effective bactericide.

Stool from patients with inflammatory bowel disease showed increased MPO concentrations depending on the stage of the inflammation.

Indications

• Marker for inflammatory activities in the gastrointestinal tract • Oxidative stress (serum)• Kidney transplant rejection (urine)• To differentiate between allergic and infect related asthma

(bronchial lavage, respiratory condensate, sputum)

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2. Radical generating and degrading enzymes

Reference Values MPO

Stool < 2000 ng/ml

Serum mean value 340 ng/ml (SD 176.7)

EDTA-Plasma mean value 98.31 ng/ml (SD 62.9)


specimen

Determination of MPO in various matrices

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ReferencesZelzer S et al. (2009) Transpl Immunol 20(3):121-6Bobbert P et al. (2008) Diabetes Res Clin Pract 82(2):179-84Dednam M et al. (2008) Clin Chem 54(1):223-5Kaschina E et al. (2008) Circulation 118(24):2523-32Klebanoff et al. (1976) Cancer Enzymology 267-285Oremek et al. (1995) MTA 4: 273-278Saiki (1998) Kurume Med J 45: 69-73

MutaGEL® OxStress II(Superoxid Dismutase [SOD2] + Catalase)

-> see Chapter 5 "Genetic Predispositions", Page 28

2. Radical generating and degrading enzymes 2. Radical generating and degrading enzymes

Matrix Plasma, SerumSample volume 100 µl (Pl.); 25 µl (Ser.) Test principle ELISA Cat. No. K 6631B

IDK® MPO

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3. AntioxidantsIt is of note that beside the above described specifi c enzymes that are involved in the degradation of ROS without being altered themselves, there are a number of naturally occurring small molecules that are able to reduce oxidized molecules in humans and animals. These small molecules often cannot be synthesized in the human body and thus need to be part of a balanced nutrition. These mo-lecules are vitamins. Vitamin defi ciency may cause oxidative stress and hence, their level needs to be in a certain optimal window. Some vitamins become toxic at high concentrations. Therefore, it is essential to be able to measure key vita-mins which act as antioxidants.

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Haptoglobin Diabetes Mellitus (DM) has become one of the most prevalent and costly chronic diseases worldwide. The global burden has risen from 30 to 382 million patients bet-ween 1985 and 2014 and current trends suggest that it will only continue to rise (glo-bal forecast for 2035: 592 million DM patients!) (Leon et al., 2015). In Germany alone, about 20 % of all statuary health insurance expenses are allocated to the treatment of diabetes and its secondary diseases.

Despite all improvements in the management of the primary risk factors hyper-glycemia and dyslipidemia as well as life style modifications, the life expectancy of DM patients is still substantially reduced. Coronary vascular diseases (CVD) represent the predominant cause of morbidity and premature mortality in diabetic patients. Therefore the primary goal of diabetes treatment should be a reduction of the risk of cardiovascular complications.

Haptoglobin is an abundant acute-phase protein. Its major function, the neutrali-zation of free haemoglobin, is essential for the prevention of oxidative tissue dam-age. Due to a polymorphism in the Hp gene, there are three different genotypes in humans. Multiple clinical studies clearly documented: DM patients with the Hp 2-2 genotype (in Europe 1/3 of all diabetics) have a 2-3-fold increased risk for cardiovas-cular events as compared to Hp 1-1 und Hp 2-2 DM individuals (a correlation which was not observed in control groups without DM) (Cahill et al., 2015; Vardi et al. 2012).

A vitamin E supplementation reduces the cardiovascular mortality in Hp 2-2 DM patients: In several clinical studies it has been shown that high dose vitamin E supplementation in Hp 2-2 DM patients can reduce the risk for myocardial infarction and cardiovascular death of up to 50 % (Ashleh et al., 2003; Blum et al., 2010; Milman et al., 2008; Costacou et al., 2012). The physiological reason might be an improve-ment of the HDL function. Reliable Hp typing is vital, as only DM patients of the Hp 2-2 genotype may benefit from a therapy with vitamin E.

References Leon BM et al. (2015) World J Diabetes 6 (13): 1246-1258Cahill LE (2015) J Am Coll Cardiol 66 (16): 1791–1799 Vardi M et al. (2012) European Journal of Internal Medicine 23: 628–632Asleh R et al. (2003) Circ Res 92: 1193-1200 Blum S et al. (2010) Pharmacogenomics 11 (5): 675 684 Milman U et al. (2008) Arterioscler Thromb Vasc Biol 28:341-347Levy AP et al. (2013) Clin Chem Lab Med 51(8): 1615–1622Costacou T et al. (2012) J Cardovasc Trans Res 5: 423 435

3. Antioxidants

Haptoglobin Typing

Sample volume 15 µlMatrix Serum, PlasmaTest principle ELISACat. No. KSA71001

The first ELISA suitable for routine determination of the Hp phenotype

not available in Portugal and Spain

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Vitamin C (Ascorbic acid)Vitamin C (ascorbic acid), being a part of the antioxidative defense system, is found in both the cytosol and extracellular spaces. Depending on the concentration and the availability of transitional metals, it has both antioxidative and prooxidative features. The antioxidative effect dominates, especially in extracellular space. Since it acts through formation of semi-dehydro-ascorbate and dehydro-ascorbate respec-tively, as an electron donor transferring hydrogen to acceptor substances by reversi-bility, ascorbic acid has strong reducing effects.

Vitamin Cmakes a contribution to the antioxidative defense system in two diffe-rent ways. On the one hand, it reacts with the reactive oxygen species (ROS), espe-cially peroxide radicals. On the other hand, ascorbic acid regenerates a-tocopherol (vitamin E). Vitamin C also has a pro-oxidative effect in combination with transition metals. It catalyses the reduction of Fe3+ to Fe2+. The created bivalent iron ions react faster with H2O2. Therefore, the formation of OH•-radicals is supported through the Haber-Weiss-Reaction.

Due to the very small concentration of free transition metals in biological tissues, the antioxidative features are predominant. As a result of increased oxidative stress, the level of vitamin C is reduced in various syndromes, e.g. the level of vitamin C in blood from HIV positive patients is significantly lower. The content in blood plasma falls from 75.7 µmol/l to 40.7 µmol/l. Smokingcauses a high consumption of vitamin C in the blood plasma. Protein thiols are oxidised and after the Vitamin C pool has been depleted, lipid peroxidation begins.

In serum and plasma vitamin C is found as ascorbic acid as well as the oxidized form: dehydro-ascorbate. Both forms are biologically active. In our vitamin C assay an oxidation is induced prior to the determination of the analyte so that both forms are measured.A dose response curve of the absorbance unit (optical density, OD at 492 nm) vs. concentration is generated, using the values obtained from standard. The concentration of the patient sample is determined directly from the linear standard curve.

Indications

• Determination of vitamin C status• Monitoring infusion therapy• Monitoring of oral vitamin C substitution (checking the individual capacity of

gastrointestinal vitamin C resorption)

Determination of Vitamin C with Colorimetric MTP-assay

Please note: Samples should be kept cool and light-protected. Samples can be measured within 24 hours after blood withdrawal.

3. Antioxidants

Available as colorimetric MTP-Assay

or HPLC-Kit

Colorimetric MTP-Assay:

Determination without HPLC

Automation is possible

Vitamin C

Sample volume 200 µlMatrix Li-Heparinate plasma, Serum,

UrineTest principle Colorimetric MTP-AssayCat. No. K 4000

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Determination of Vitamin C with HPLC-Kit

ReferencesAllard et al. (1998) Am J Clin Nutr 67:143-147Balz et al. (1991) Biochem J 277:133-138

3. Antioxidants 3. Antioxidants

Reference value Vitamin C

Li-Heparinate plasma 4 – 15 mg/l

HPLC-Kit:


Stable controls

Mobile phase can be circulated

Continuous external quality control by interlaboratory tests of e.g. INSTAND e.V.: Evaluation "BRAVO" for "Vitamins" in July 2016

Vitamin C (HPLC) Flow rate 0.75 ml/minUV-Detector 254 nmSample volume 200 µlElution isocraticMatrix Li-Heparinate plasmaTests 100 Cat. No. KC2900

Reference Values Vitamin C

Li-Heparinate plasma: 4 – 20 mg/l

Chromatogram Vitamin C

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Vitamin A/E (Retinol/Tocopherol)Vitamin E (Tocopherol) belongs to the intracellular, non- enzymatic, hydrophobic antioxidants. It is part of the intracellular biomembrane and there it primarily pre-vents lipid peroxidation. Due to its localisation in the membranes, it reacts with O2, O2

•- and lipid peroxyradicals in particular. Each molecule can react with two lipid per-oxide radicals. A resonance-stablilized a-tocopherol radical is generated in the first step and the stable a-Tocopherol-chinon is created in the second step. a-tocopherol radicals can diffuse through membranes and lipid surfaces. Thus they can be regene-rated by ubiquinone, and ascorbic acid and glutathione.

Indications

• Disturbed lipid resorption • Cystic pancreatitis • Disturbance of bone growth • Visual disturbance • Alteration of retina • Degeneration of testes and ovaries • Reduction of heart attack risk

3. Antioxidants

Simple sample preparation(only a precipitation step)

Internal standard

Total running time <15 min

Continuous external quality control by

interlaboratory tests of e.g. INSTAND e.V.:

Reference Values Vitamins A / E

Vitamin A (Serum) 200 – 800 µg/l

Vitamin E (Serum) 3 – 14 mg/l

Flow rate 0.8 ml/minlUV-Detection Vitamin A: 325 nm; Vitamin E: 300 nmSample volume 250 µlMatrix Serum, PlasmaElution isocraticTests 100 Cat. No. KC1600

Vitamin A/E (HPLC)

Chromatogram Vitamin A / E

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Ubiquinone / Coenzyme Q10 Ubiquinone was first isolated in the 50's by Prof. Green's group (Wisconsin). The function was investigated by Prof. Mitchel who received the Nobel price for his research on the oxydative phosphorylation pathway.

Ubiquinone is a coenzyme which is represented in every cell and the whole me-tabolism. It is made up of a chinonic ring and an isoprenic sidechain. In humans ubi-quinone can be synthesized and absorbed through nutrition. Ubiquinone has two different pysiological functions:

• Component of the energy metabolism • Radical scavanger

During the reduction of oxygen in the oxydative phosphorylation 3 Mol ATP is generated. In this reduction, electrons are tranferred from NADPH to oxygen via 6 different redox systems. Ubiquinone is the least abundant redox system in the membrane of the mitochondria. Because of the low amount, it is the speed control-ling redox-system in the energy metabolism. Normally the amount of ubiquinone is sufficient, but with growing age and exposure to sunlight it is reduced to 50 %. Patients under treatment with cholesterine reducers show a dose related decrease of Coenzyme Q10.

Ubiquinone has a high amount of carbon doublebonds and therefore a higher potential of reduction than vitamin C or vitamin E. Therefore it is the first line of defense against free radicals. Thus ubiquinone is an optimal stabilizer of the ion channels of the membranes.

Indications

• Determination of Coenzyme Q10/ Ubiquinone status (especially under treatment with cholesterine reducers as stains) • Cardiovascular disease • Carcinogenesis • Aging

ReferencesMortensen SA et al. (1997) Molec Aspects Med 18:137-144

3. Antioxidants 3. Antioxidants

First commercial obtainable kit for the determination of Ubiquinone

Internal standard runs in the chromatogram after Q10 ; therefore no false-negative results

Reference Values Ubiquinone / Coenzyme Q10

EDTA blood / EDTA plasma 0.67 – 0.99 µg/ml

Ubiquinone (HPLC) Flow rate 0.8 – 1.2 ml/minDetection UV: 275 nmSample volume 100 µlElution isocraticMatrix EDTA-blood; EDTA-plasma, SerumTests 100 Cat. No. KC1700

Chromatogram Ubiquinone / Coenzeyme Q10

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Glutathione (GSH/GSSG)Glutathione is the most important cellular protection system against the toxic effects of metals such as Hg2+ and MeHg. It is a tri-peptide of cysteine, glycine and glutamic acid. As an antioxidant, it can catch free radicals, reduce H2O2 and stabilize sulf-hydryl groups. H2O2 is transformed into oxygen and water with the aid of GSH-peroxidase, whereby GSH itself is oxidised to GSSG. The enzyme glutathione reductase reduces GSSG to active GSH.

Glutathione is transferred to xenobiotica via the enzyme glutathione-S-transferase. The water solubility of this substance is increased and the elimination facilitated.

Intracellular redox regulation is impaired by these xenobiotics. The result is a com-petition between the excretion of GSSG and the GSH-xenobiotica-complex. This is the reason for the intracellular increase of the GSSG concentration. In addition, chlorpy-rifos can block the reduction of GSSG, thus shifting the balance of GSH to GSSG. Due to this shift, cell functions of all organs are extremely affected and vitally threatened.

Patients suffering from chronic lymphatic oedema show a 20% lower GSH level in their erythrocytes compared to controls (1.68 mmol/l cells compared to 2.04 mmol/l). The GSSG concentration, however, is increased by about 5% (59.25 µmol/l cells com-pared to 56.79 µmol/l).

Indications

• Oxidation status (e.g. in diabetes)• Studies of oxidative stress in the cell• Stress through increased oxygen turnover under high performance conditions.

ReferencesBeier et al. (1994) Lymphol 18: 8-11

3. Antioxidants

HO

NH2

O O O

OHO

NH

S

NH

HO

OOO

OH NHNH2

S

O

HN

Glutathione (oxidized) GSSG Glutathione (reduced) GSH

HO

NH2

SHO O O

OH

HN

ONH

Determination of the oxidized (GSSG) and the

reduced (GSH) form of glutathione

Internal standard

Mobile phase can be recirculated

Reference Values Glutathione

EDTA-whole blood

total: 763 – 1191 µmol/l

reduced: 620 – 970 µmol/l

Chromatogram GSH

GSH/GSSG (HPLC) Flow rate 0.75 – 1.0 ml/minDetection Fluorescence: Ex.: 385 nm; Em.: 515 nmSample volume 100 µlElution isocraticMatrix EDTA whole bloodTests 100 Cat. No. KC1800

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3. Antioxidants

4. Modified molecules (lipids, proteins, sugar moieties, DNA)

If oxidative stress is present, cell damage occurs. Oxidation targets are lipids, proteins, sugars, and the DNA. The resulting damage to the cell /living organism and thus the resulting disease state is highly dependent on the specific target. For example oxidation of the DNA may cause mutations within the genome. Oxidation of proteins such as enzymes or receptors usually causes a loss of func-tion of the specific enzyme/receptor. Oxidation of sugars leads to products that for their part, are toxic to cells. Oxidative stress in disease status is never uniform. It is therefore important to identify the targets of oxidative stress in order to be able to understand the pathogenesis of a certain disease and to interfere speci-fically. Thus it is a great achievement that there is now a broad spectrum of test systems available to analyse the targets of oxidation in detail.

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Oxidized LDL (ox-LDL) / MDA AdductLipid peroxidation is a natural process essential for cell growth. However, when the oxidative stress overwhelms the antioxidative cell defense, the balance is disturbed and enhanced formation of lipid peroxidation products occurs. At present, lipid peroxidation is considered to be one of the basic mechanisms involved in the initia-tion and progression of many diseases. Various studies have provided evidence that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease.

Lipid peroxidation products are formed during normal cell metabolism via the production of an excess of free radicals that can react with unsaturated fatty acids , in particular low-density lipoprotein (LDL), the major carrier of plasma chole-sterol. LDL is eliminated by macrophages. Normally, receptor-mediated uptake of LDL is suppressed through down-regulation of LDL receptor expression in response to increasing cholesterol levels. Once LDL is oxidized, it is still internalized by macro-phages but through scavenger receptors whose expression is not controlled by cholesterol loading. The binding of oxidized LDL (oxLDL) is the step by which chole-sterol accumulation in macrophages is induced, transforming them into lipid-loaded ‘foam cells’. This process is accompanied by extensive cell proliferation and elabo-ration of extracellular matrix components and contributes to the genesis and pro-gression of atherosclerosis by promoting endothelial damage and amplifying the inflammatory response within the vessel wall. Cholesterol loaded macrophage ‘foam cells’ are present in the earliest detectable atherosclerotic lesions, the precursor of more complex atherosclerosis that cause stenosis and limited blood flow. These ad-vanced lesions ultimately represent the sites of thrombosis leading to myocardial infarction.

The oxLDL ELISA is intended for the quantitative determination of oxLDL in EDTA-plasma and serum. The test recognizes MDA-modified Apolipoprotein B 100, contai-ning less than 60 MDA units per molecule.

Scientific study of Koubaa et al. (2007)

Using the Immundiagnostik’s ELISA Kit, a mean value of 95,32 ± 37,85ng ox-LDL/ml was estimated for healthy control subjects (n=120) in serum/plasma. Furthermore, the obtained results demonstrate that a significantly elevated oxLDL concentration (142,37 ± 49,84 ng oxLDL/ml) was found in Typ-2 Diabetes patients (n=86) compared with healthy controls.In addition, higher oxLDL values were detected in Typ-2 Diabetes patients with hypertension, as compared with diabetic patients without hypertension.The results of the study are summarized in the following tables.

Sample ox-LDL [ng/ml]

Typ-2 Diabetes patients without hypertension

111,16 ± 33,42

Typ-2 Diabetes patients with hypertension

157,4 ± 49,9

Sample ox-LDL [ng/ml]

Controls, healthy (n=120) 95,32 ± 37,85

Typ-2 Diabetes patients (n=86)

142,37 ± 49,84


Measurement of oxidized LDL as a marker

for lipid peroxidation

Our ox-LDL/MDA Adduct ELISA Kit recognizes MDA-modified Apolipoprotein B

100, containing less than 60 MDA units per molecule

“ …the new oxLDL ELISA (Immundiagnostik) is more

sensitive … this assay but not the competitive method

confirmed the expected efficacy of PPARg-activation

in decreasing oxidized LDL particles in the observed

patient population. “

Pfützner A et al (2005) 5th Diabetes Technology Meeting, San Francisco

(10.-12. November 2005)

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Indications

• Monitoring oxidative stress (e.g. in hemodialyzed patients)• Inflammatory processes

References Kurban S et al. (2010) Clin Biochem 43(3):287-90.Börekçi B et al. (2009) Turk J Med Sci 39 (2): 191-195Pfützner A et al. (2009) Clin Lab (7+8)55:275-81Göçmen AY et al. (2008) Clin Biochem 41(10-11):836-40.Koubaa N et al. (2007) Clin Biochem 40(13-14):1007-14.Licastro F et al. (2007) Arch Gerontol Geriatr 44 Suppl 1:225-32Pfützner A et al (2005) Clinical Evaluation of a new ELISA method for determination of oxidized LDL parti-cles – a potential marker for arterisclerotic risk in diabetes mellitus. Abstract of the 5th Diabetes Technolo-gy Meeting, San Francisco (10.-12. November 2005)Holvoet P et al. (2001) Arterioscler Thromb Vasc Biol 21(5):844-8Witztum JL et al. (1997) Ann N Y Acad Sci 811:88-96

4. Modified molecules (lipids, proteins, sugar moieties, DNA) 4. Modified molecules (lipids, proteins, sugar moieties, DNA)

Matrix Serum, EDTA Plasma, dried blood

Sample volume 10 µl (Ser., EDTA pl.) 50 µl (dried blood)Test principle ELISA Cat. No. K 7810

ox-LDL/MDA Adduct

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Autoantibodies against oxidized LDL (anti ox-LDL)Due to their double bond, unsaturated fatty acids are especially prone to oxidants. Peroxidation of cell membrane lipids lead to a total functional loss of the cell. Peroxi-dation of lipid molecules of the LDL-particle (Low Density Lipoprotein) is an essential trigger of arteriosclerotic vascular aging. An elevated risk for arteriosclerosis is the consequence.

Oxidized LDL is immunogenic and specific autoantibodies against its epitopes (e.g. malondialdehyde-lysine) can be detected in serum. These antibodies detect tissue from arteriosclerotic lesions in contrast to tissues from normal arteries.

Indications

• Arteriosclerosis, risk assessment of heart attack and stroke • Detection of autoantibodies against oxidized LDL

ReferencesSalonen JT et al. (1992) Lancet 339:883-887Esterbauer H et al. (1991) Free Radical biol Med 13:341-390Tatzber F et al. (1991) Arteriosclerosis 89:203-208


Detection of Autoantibodies against oxidized LDL

Use of Cu2+-oxidized LDL

Short incubation times (<3 h)

anti-ox-LDL

Sample volume 50 µlMatrix EDTA-Plasma, SerumTest principle ELISACat. No. K 7809

21

Malondialdehyde (MDA)As a result of the oxidation of unsaturated fatty acids of the cell membranes, a wide spectrum of various hydroperoxides can be produced in the organism. These fatty acid derivatives are chemically unstable and are quickly transformed into aldehydes. Amongst these is malondialdehyde, which has the property of being able to cross-link proteins and lipids.

The MDA levels in the serum of patients with terminal kidney failure (dialysis) (4.05 + 1.7 µmol/l), with septic shock (3.4 + 2.3 µmol/l), with a high-risk pregnancy (9.4 + 4.6 µmol/l) as well as female volunteers taking contraceptives (8.37 + 3.57 µmol/l) are significantly increased compared to baseline values (1.97 + 0.41 µmol/l).

A comparison of patients with “unstable angina (UA)“ and patients with myocardi-al infarction (MI) shows that the MDA level in the plasma of patients with UA incre-ases until the 5th day and then decreases until the 12th day. An immediate increase of the concentration can be seen in MI patients, followed by a decrease within the following 12 days.

Indications • Indicator for lipid peroxidation. Useful for biological, medical and nutritional

studies • Biochemical marker for oxidative stress

ReferencesErol B et al. (2010) Fertil Steril 93(1):280-2Tasoulis MK et al. (2009) Tohoku J Exp Med 219(3):193-9Loeper et al. (1991) Free Rad. Res. Comms., Vols. 12-13,: 675-680Nielsen F et al. (1997) Clin Chem 43: 1209-1214


Chromatogram MDA

O O

Malondialdehyde

Marker for oxidative damage

5 minutes run time

Reference Values MDA

EDTA-plasma: < 1 µmol/l

MDA (HPLC) Flow rate 0.8 – 1.0 ml/minDetecion Fluorescence: Ex: 532 nm, Em. 550 nmSample volume 20 µlElution isocraticMatrix Plasma, Serum, UrineTests 100 Cat. No. KC 1900

22

Carbonyl ProteinReactive oxygen species (ROS) can oxidize proteins, lipids, and DNA, causing dama-ge of their structure and function and cell injury. Proteins are oxidized by free radi-cals, whereby the constituent amino acids are variously modified or degraded. The modifications result in new functional groups such as carbonyl groups , which may lead to protein fragmentation, formation of protein-protein cross-linkages, disrupti-on of the tertiary structure and loss of functional activity. In addition, ROS are directly associated with diseases like atherosclerosis, rheumatoid arthritis, Alzheimer’s and Parkinson’s disease as well as ageing and cancerogenesis. Carbonyl proteins are formed by a variety of oxidative mechanisms and are sensitive indices of oxidative injury. The quantity of protein carbonyls in a protein sample can be determined by derivatizing with dinitrophenylhydrazine (DNPH) and measuring the bound anti-DNPH antibodies. The ELISA method enables carbonyls to be measured quantitatively with picogram quantities of protein.

Suitability of the Assay• EDTA plasma, • bronchioalveolar lavage,• cerebrospinal liquid,• cell extracts,• other soluble protein containing liquids.

Indications • Arterosclerosis • Alzheimer’s disease

• Parkinson’s disease• Rheumatoid arthritis• Uraemia• Diabetes• Ageing• Cancerogenesis

ReferencesTrudel S et al. (2009) PLoS ONE 4(6): e6075. doi:10.1371/journal.pone.0006075Taieb MAE et al. (2009) Eur J Obstet Gynecol Reprod Biol 144 Suppl 1:S199-203Matzi V et al. (2007) Eur J Cardiothorac Surg 32(5):776-82Dalle-Donne I et al. (2006) Clin Chem. 52(4):601-23. Epub 2006 Feb 16. ReviewDalle-Donne I et al. (2006) J Cell Mol Med 10(2):389-406. ReviewDalle-Donne I et al. (2003) Clin Chim Acta 329(1-2):23-38 Stadtman ER, Oliver CN (1991) J Biol Chem 266(4):2005-8. Review


Ultra sensitive assay for the determination of carbonylated

proteins now available

Also suitable for samples poor in proteint!

4 µl sample volume only

Test procedure 6 hours only

Sample volume 4 µlMatrix Plasma , intra-/extracellular liquidsTest principle ELISA Cat. No. KR7822

Carbonyl Protein*

Sample volume 10 µlMatrix Serum, PlasmaTest principle ELISA Cat. No. K 7870

Carbonyl Protein

* For Research Use only

23

AOPP

AOPP (advanced oxidation protein products) reflect the protein oxidative damage that occurs in patients with oxidative stress.

Haemodialysis patients combine a massive generation of reactive oxygen species at each dialysis session with a chronic deficiency in the major antioxidant system. The measurement of AOPP is proposed to be a reliable marker to estimate the degree of oxidant mediated protein damage in uremic patients and to predict the potential efficiency of therapeutic strategies aimed at reducing such an oxidative stress.

Our colorimetric test is available as a micro titer plate assay or for cuvettes.

Research areas: • Monitoring oxidative stress (e.g. in hemodialyzed patients) • Inflammatory processes

* For research use only


ReferencesCorsi-Romanelli MM et al. (2005) Advanced Oxidation Protein Products (AOPP) per Monitorare lo Stress Ossidativo ed un possibile stato inflammatorio cronico in un gruppo eterogeno per eta' die soggetti con trisomia 21. Poster presented at SIMeL Congress, November 2005, TriesteDeschamps-Latscha B et al. (2005) Am J Kidney Dis 45(1):39-47Deschamps-Latscha B et al. (2004) Kidney International 66: 1606-1612Nguyen-Khoa T et al. (2001) Nephrol Dial Transplant 16: 335-340Witko-Sarsat V et al. (1996) Kidney International 49: 1304-1313


AOPP (MTP assay) *

Sample volume 125 µlMatrix EDTA-PlasmaTest principle photometricCat. No. K 7811w

AOPP (cuvettes) *

Sample volume 400 µlMatrix EDTA-Plasma Test principle photometricCat. No. K 7811c

Only oxidized proteins are determined

Ready-to-use-buffer for diluting samples and standards

Lyophilized thermostable standard

Processing small amounts of specimen

MTP assay Yields quick results

Sample volume: 125 µl

24

Nitrotyrosine

Nitrotyrosine is the nitrated form of the amino acid tyrosine. The accumulation of protein bound nitrotyrosine is associated with cardiovascular diseases that are based on inflammatory processes (e.g., atherosclerosis, myocardial infarction, diabetic vas-culopathy, hypertension, or coronary heart diseases). A growing number of studies have also associated the accumulation of nitrotyrosine with neurological diseases (Alzheimer´s disease, Parkinson´s disease, multiple sclerosis, stroke). With treatment of some of the associated diseases the levels of nitrated tyrosines have been shown to decrease, so nitrotyrosine has been stated to be a marker of nitrosative stress.

During inflammatory processes, large amounts of nitric oxide (•NO) are locally released from L-arginine. This reaction is catalyzed by the enzyme NO-synthase (NOS). Other causes for the increased •NO production are exposure to chemicals or heavy metals, drugs, nicotine, or physical and psychological stress, as well as extraor-dinary physical strain with increased oxygen consumption.

In high concentrations, •NO that is not trapped by mitochondrial superoxide dismutase (SOD) reacts with superoxide (O2•–) to form peroxynitrite (ONOO–). Peroxynitrite is implicated as a key oxidant species in several pathologies and is known to be cytotoxic (nitrosative stress). Peroxinitrite is highly reactive and shows a high affinity to aromatic amino acids, e.g., to the phenolic ring of tyrosine. The nitra-tion of tyrosine in general is a natural process within the post-translational protein modification. Nitrotyrosine is a stable product and might be seen as a correlate of peroxynitrite production, and its accumulation in cells and tissues is a marker of oxi-dative stress and nitrosative stress, respectively (Ischiropoulos 2008).

Indications • Cardiovascular diseases • Neurological diseases • Thyroid disturbances • Blockade of biochemical pathways • Mitochondriopathy

ReferencesGonsette RE (2008) J Neurol Sci, Vol 274, Issue 1-2, 48-53Ischiropoulos H (2008) Arch Biochem Biophys Oct 30Peluffe G, Radi R (2007) Cardiovasc Res. Jul 15 : 75(2) :291-302Souza JM et al. (2008) Free Radic Biol Med. Aug 15 ; 45 (4) :357-356


Nitrotyrosine

Matrix Stool, EDTA plasma, SerumSample volume 100 mg (Stool); 50 µl (EDTA plasma, Serum)Test principle ELISA Cat. No. K 7824

Nitrotyrosine

Matrix EDTA Plasma, Serum, dried blood

Sample volume 15 µl (EDTA Pl., Serum) 50 µl (dried blood)Test principle ELISA Cat. No. K 7829

8-OHdG(8-Hydroxydesoxyguanosin)

Approximately 5 % of inhaled oxygen is estimated to be converted to reactive oxygen species (ROS) in vivo. These molecules attack proteins, DNA and lipids. The DNA-base guanine is altered to the modified form 8-hydroxydesoxyguanosin (8-OHdG). A re-pair mechanism changes the altered nucleoside with the modified guanine thereby releasing 8-OHdG. The free 8-OHdG is transported via circulation to the kidney where it is excreted with the urine. If anti-oxidative protection is not sufficient or weakened ROS-attacks can trigger aging processes and pathogenesis of diseases.8-OHdG is produced by the reaction of hydroxy radicals with the DNA and is a good biomarker for the extension of oxidative stress at the cellular level. The administration of acetylcysteine can counteract an intracellular depletion of anti oxidants.

Our competitive ELISA for the determination of the oxidated DNA product 8-OHdG is based on highly specific monoclonal antibodies.

Research areas:• Determination of the intracellular oxidative stress level• Monitoring of an anti oxidative therapy in humans and animals• Monitoring of changes in the oxidative status in response to sports or life style

changes

ReferencesToyokuni et al. (1997) Lab Invest 76:365 – 374.Sri Kantha et al. (1996) Biochem Biophys Res Comm 223:278 – 282.Erhola et al. (1997) FEBS Lett 409:287 – 291.

25


Easy handling

Mikrotiter plate test

Determination in urine

High-sensitive version for determination in serum and plasma

Reference range 8-OHDG

Urine 2 - 20 ng/ml

8-OHDG (ELISA)*

Sample volume 100 µlMatrix Serum, tissueTest princple ELISACat. No. KJKOGHS10E

8-OHDG*

Sample volume 100 µlMatrix UrineTest principle ELISACat. No. KA4000

*For Research use only!


5. Genetic predispositionsGenetic variants, largely in form of single nucleotide polymorphisms (SNPs) are found in almost every human gene. Considering oxidative stress, many poten-tially significant genetic variants do exist. Examples include glutathione trans-ferase null alleles, a low-activity variant of quinone reductase 1, altered heme oxygenase, nitric oxide synthase, paraoxonase, and mitochondrial superoxide dismutase. The actual consequence of a base change can, of course, impinge on gene transcription, mRNA stability, or protein function. Detrimental amino acid changes and altered gene regulation have been the focus of most attention so far.

Basic and clinical research on human genetic variation in oxidative stress–rela-ted genes demonstrated an association between SNPs and disease state. Fur-thermore, the effect of antioxidants on expression levels needs to be determined when interpreting genetic data on oxidative stress genes in relationship to a given phenotype. When coupled to indices of oxidative stress such as oxidized DNA, lipids, and proteins, the genetic variants can provide mechanistic insight into defense systems of the human body.

26

27

MutaGEL® OxStress I(Endothelial NO-Synthase (eNOS) + NAD(P)H Oxidase)

Oxidative Stress contributes to the pathogenesis of coronary heart disease (CHD) based on the development of atherosclerosis in coronary arteries. Endothelial Nitric Oxide Synthase (eNOS) and NAD(P)H oxidase in the vessel wall act as im-portant superoxide-generating system in the vasculature. Therefore, shear stress- induced endothelial nitric oxide (NO) release as well as a reduced NAD(P)H-oxidase function helps to retard atherosclerosis. Functionally relevant polymorphisms in eNOS gene and NAD(P)H-oxidase gene are analyzed by this kit MutaGEL® OxStress I.

5. Genetic predispositions

Sample volume 200 µlMatrix DNA (e.g. whole blood, cheek

swab)Test principle PCR (RFLP)Cat. No. KE09008

MutaGEL® OxStress I *

MutaGEL® OxStress II(Superoxid Dismutase [SOD2] + Catalase)

During oxidative stress the levels of several indicative molecules are influenced by some common DNA polymorphisms of enzymes, which metabolize the agonists of the radicalic or nonradicalic oxidants - mainly superoxide and hydroxide anion. The lacking reduction can lead to diabetes and to accelerated old age symptoms or even cancer development. SOD2 catalyzes the biochemical reaction from superoxide radi-cals (from different metabolic pathways) to H2O2. Catalase transforms subsequently this H2O2 to H2O and O2.

The SOD2-polymorphism Val16Ala alters the recognizing sequence for mito-chondria, where ROS (reactive oxygen species) are deactivated. The valine-allel is found increased in cancer patients probably due to the reduced protection for oxi-dative stress (over chronically activation of B-cells and releasing of cytokines) leading to cancerogenesis. The relative frequency for Val-Val homozygotie in lung cancer is 1,7 – in bladder cancer even more. In breast cancer – especially under unfavourable “cofounder conditions” (e. g. heavy women with long menstruation year load) also the alanin-allel is neoplastic (due to higher toxicity of metabolites).

The Catalase-polymorphism C-262T in the promoter region influences the en-zyme concentration in the erythrocytes (each T-allel increases the catalase activity). An influence of these genetic variants is described for breast and pancreas cancer (CC-homocygotie is breast cancer protective) and for neuropathies of diabetic pati-ents (here CC is pathogen). Additonal influence on oxstress reduction activity have e. g. the complete deletion of glutathion-S-transferase genes M1 and T1 (see “Muta-GEL® GST-M1/T1”) as well as sequence variation of endothelial nitrogen oxide syn-thetase (eNOS) and NAD(P)H oxidase (see “MutaGEL® Oxstress I”).

28


-> see Chapter 2 "Radical generating and degrading enzymes", Page 9


MutaGEL® OxStress II (SOD2/Catalase)

Sample volume 200 µlMatrix DNA (e.g. whole blood, cheek

swab)Test principle PCR (RFLP)Cat. No. KE09011

MutaCHIP® TOXO(Determination of detoxification risk factors)

The analysis of a gene panel that is associated with detoxifying enzymes can deter-mine individual detoxification risk factors that play an important role in the degradati-on of exogenic metabolites (e.g. chemicals, dyes, environmental toxins, aromatic com-pounds from tobacco or barbecued food, hormone replacement substances, etc.).

The individual genotype is analysed directly with the MutaCHIP® reader and displayed along with a detailed report and a therapy recommendation.

MutaCHIP® TOXO covers the analysis of polymorphisms (SNPs) in the following genes: CYP1A1*2A, CYP1A2*1C, CYP1A2*1F, CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2C19*17, CYP3A5*2, CYP3A5*3, CYP3A4*1B, CYP3A4*2, CYP3A4*3, CYP3A4*17,GST M1 (14kb deletion), GST T1 (50kb deletion), GST P1 (Ile105Val, Ala-114Val), MDR-1 (3435C>T), COMT (Val158Met), CYBA (242C>T), NAT2 (C481T, G590A, G857A).

29

5. Genetic predispositions 5. Genetic predispositions

MutaCHIP® TOXOSample volume 200 µlMatrix DNA (e.g. whole blood, cheek

swab)Test principle Gene Chip with MAAT-reader systemCat. No. KF390011

MutaPLATE® GST-M1/T1 MutaPLATE® GST-P1

Glutathione-S-transferase deficiency can be genetically determined: more than 50% of Caucasians do not carry the GST-M1 gene and about 20% do not have the GST-T1 gene. Our kit “MutaPLATE® GST-M1/T1” allows the simultaneous detection of the glutathione-S-transferase-genes GST-M1 and GST-T1 (and additionally the albumin gene as internal control).

The GST-P1 gene compensates (at least in part) for the frequent deletion of GST-M1- or GST-T1-genes in Caucasians (about 50 % respectively 20 %). The kit “MutaPLATE® GST-P1” allows the detection of the glutathione-S-transferase-genes GST-P1 encoding for the enzyme glutathione-S-transferase P1.

ReferencesSundberg et al. (1994) Nephron 66:162-169Hayes et al. (1989) Proceedings of 3rd International GST Conference, Edinburgh, Scotland Sundberg et al. (1994) Nephron 67:308-316

30


Matrix DNA e.g. whole blood, cheek swab)

Sample volume 200 µlTest principle real time PCR (open systems) Cat. No. KF1902196

MutaPLATE® GST-M1/T1 (TM)

Matrix DNA e.g. whole blood, cheek swab)

Sample volume 200 µlTest principle real time PCR (open systems)

Cat. No. KF1903196

MutaPLATE® GST-P1 (TM)

ADMA / ADMA Xpress Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase, an enzyme that catalyses the production of nitric oxide from the amino acid arginine. Nitric oxide (NO) is an endothelium-derived vasoactive mediator and is involved in the modulation of blood flow and blood pressure. Inhibition of NO production im-pairs endothelial function – this is why NO has been described as an endogenous anti-arteriosclerotic molecule. Accordingly, elevated circulating ADMA levels are dis-cussed tocontribute essentially to the pathogenesis of arteriosclerosis. In dialysis pa-tients, for example, the degree of arteriosclerosis correlates significantly with eleva-ted circulating ADMA. ADMA was found to predict cardiovascular risk independently of other variables and is hence an important prognostic factor in patients with arteri-osclerosis, coronary artery disease, peripheral arterial disease, hypertension, chronic heart failure, hypercholesterolemia, chronic kidney disease, or diabetes mellitus.

Indications• Coronary heart disease• Chronic kidney disease and dialysis• Peripheral arterial disease• Chronic heart failure• Hypertension• Diabetes mellitus• Hyperlipoproteinaemia• Preeclampsia• Erectile dysfunction

31


6. Additional biomarkers of oxidative stressImmundiagnostik offers a variety of other parameter for laboratory diagnostics that are directly or indirectly related with oxidative stress and that can serve as indicators for the evaluation of disease status.

Competitive ELISA in microtiter plate formate

Excellent correlation with LC/MS and with automated tests

Good linearity in the range of 0.1 µM - 2 µM

No false-positive values due to calibrators and controls which are based on ADMA-free plasma!

No cross-reactivity with L-arginine, SDMA and N-monomethyl-arginine

ADMA

Sample volume 50 µlMatrix EDTA plasma, Citrate Plasma, SerumTest principle ELISACat. No. K 7828

ADMA Xpress

Sample volume 25 µlMatrix Serum, EDTA-PlasmaTest prinicple ELISACat. No. K 7860

ADMA Xpress ELISA:• Only 3 hours running time• temperature robust• automatable• suited for small sample volumes

ReferencesCelik M et al. (2014) J Diabetes Res. 2014: 139215Kurtoglu E et al. (2014) Angiology 65 (9) (Oct): 788–93Watarai R et al. (2014) J Epidemiol Community Health 24 (3): 250-257Brenner T et al. (2012) Mediators Inflamm. 2012: 210454.Ayer JG et al. (2009) Arterioscler Thromb Vasc Biol 29(6):943-9Ayer JG et al. (2009) Am J Clin Nutr 90(2):438-46Puchau B et al. (2009) Metabolism 58(10):1483-8Hohberg C et al. (2008) Diabetes Stoffwechsel u Herz Suppl. 1: S43-S47Ott C et al. (2008) Atherosclerosis 196(2):704-11Ozgurtas T et al. (2008) Atherosclerosis 200(2):336-44Corsi MM et al. (2007) Poster P173 of the 6th World Congress on Hyperhomocysteinemia, Saarbrücken, Germany, June 5-9, 2007, CCLM 45(5)Flieser D (2005) Eur J Clinical Invest 35: 71-79Kielstein J T et al. (2005) Arterioscler Thromb Vasc Biol 25: 1-5Kielstein J T et Zoccali C (2005) Am J Kidney Dis 46: 186-202Vallance P et al. (2004) Arterioscl Thromb Vasc Biol 24: 1023-1030Böger RH (2003) Cardiovasc Res 59: 824-833Zoccali C et al. (2001) Lancet 358: 2113-2117Kielstein JT et al. (1999) J Am Soc Nephrol 10: 594 - 600

32

6. Additional biomarkers of oxidative stress

ADMA (Mouse/Rat) *

Sample volume 25 µl Matrix EDTA plasma, Serum,

Cell culture media Tests principle ELISACat. No. KR3001


ADMA *

Sample volume 20 µlMatrix UrineTest principle ELISACat. No. K 7830


SDMAThe dosage of most drugs must be adapted in renal insufficiency, making accurate assessment of renal function a prerequisite in clinical medicine. Furthermore, even a modest decline in renal function has been recognized as a cardiovascular risk.

In clinical practice serum creatinine is typically used to asses renal function, but this markerdoes not increase at modest decline in renal function. Consequently, the-re is an ongoing search for suitable endogenous markers of renal function.

Symmetric dimethylarginine (SDMA) is a methylated derivative of L-arginine which is strictly eliminated by renal extraction. Therefore, SDMA plasma level stron-gly correlates with renal function. In 18 studies with more than 2136 patients syste-mic SDMA concentrations correlated highly with inuline clearance and with vario-us clearance estimates combined, as well as with serum creatinine (Kielstein et al., 2006). These data confirm that SDMA is a sensitive and reliable marker of renal dysfunction. Moreover, increased SDMA level appear to correlate with sequential organ failure of liver and kidney and with an increased cardiovascular risk.

SDMA elevated in:

• Chronic renal insufficiency • Endstage renal diseases • Kidney transplantation • Hypertension • Diabetes mellitus • Liver failure

ReferencesKoch A et al. (2013) Inflamm: 413826Tenderenda-Banasiuk E (2013) Disease Markers 35: 407-412Kielstein JT et al. (2009) Semin Dial 22: 346–350D’Apolito O et al. (2008) Clinical Biochemistry 41:1391-1395Kielstein JT et al. (2006) Nephrol Dial Transplant 21: 2446-2451Bode-Böger SM et al. (2006) J Am Soc Nephrol 17:1128-1134Fleck C et al. (2003) Clinical Chimica Acta 336:1-12.

33

6. Additional biomarkers of oxidative stress 6. Additional biomarkers of oxidative stress

Endogenous marker for renal dysfunction

Microtiter plate ELISA

High precision

No cross reactivity with L-arginine, ADMA and N-monomethylarginine

Sample volume 50 µlMatrix EDTA plasma, SerumTest principle ELISA Cat. No. K 7780

SDMA

L-Citrulline Nitric oxide (NO) is an free-radical gas and is an important signalling molecule that acts in many tissues to regulate a diverse range of physiological processes. Since the discovery that nitric oxide is able to induce vasodilation a large number of other roles have been described for NO. It is also known to play a role in the immune system, the nervous system, in inflammation and in programmed cell death (apoptosis). NO has also been implicated in smooth muscle relaxation, pregnancy and blood vessel formation (angiogenesis). The biological effects of NO are mediated through the re-action of NO with a number of targets such as haem groups, sulfhydryl groups and iron and zinc clusters.Role of NO in the immune systemNO can be produced by a number of cells involved in immune responses. In particu-lar cytokine-activated macrophages can produce high concentrations of NO in order to kill target cells such as bacteria or tumour cells. NO-mediated cytotoxicity is often associated with the formation of nitrosyl-thiol complexes in enzymes within the tar-get cell. NO has been shown to kill cells by disrupting enzymes involved in the Kreb‘s cycle, DNA synthesis and mitochondrial function.

Role of NO in inflammationNO may act as a mediator of inflammatory processes. It enhances the effect of cyc-looxygenase (COX) and stimulates the production of pro- inflammatory eiconosoids. Furthermore, NO production can be induced, through the upregulation of iNOS, by a number of factors involved in inflammation, including interleukins, interferon-gam-ma, TNF-alpha and LPS.

L-citrulline as surrogate marker for NOMeasurement of NO production in vivo is difficult because of its short half-life and the need for specialised equipment that uses chemiluminescence detection. Ci-trulline level can be used as surrogate marker for estimating NO production.

Indication for the detection of urinary citrulline

• Estimating the NOS activity (NO production) • Detection of nitrosative stress due to an enhanced synthesis of inducible nitric

oxide (iNO)

ReferencesWanchu A (2001) J Clin Rheumatol 7 (1):10-5


34

CYTOSOL

MITOCHONDRIACitrulline

Arginine

Argininosuccinate

Ornithine

Urea

Citrulline NO cycle conversion of arginine into

citrulline, producing NO

Urea CycleRegeneration of arginine

�

�

NO

�

NOS

�

�

L-Citrulline

Sample volume 500 µlMatrix Urine, Serum, PlasmaTest principle colorimetricCat. No. K 6600

C-reactive Protein (CRP) C-reactive protein (CRP) is mainly formed in hepatocytes. The rate of its synthesis is determined by the influence of cytokines involved in the inflammatory processes. The biological half-life of the acute phase protein is estimated to be 13-16 hours.

The CRP assay proved itself as a marker for inflammation. A relationship between inflammatory reactions and cardiovasular diseases (e.g. arteriosclerosis, latent or chronically persisting infections) has been described and has also proven CRP to be an indicator of myocardial infarction. Elevated levels of CRP are also a good predictor of future cardiac diseases.

Indications • Inflammatory reactions • Therapy monitoring in infections, tissue injury and similar diseases • Prevention of coronary heart diseases

ReferencesYaturu S et al. (2006) Cytokine 34(3-4):219-23. Epub 2006 Jul 5Zwaka T P et al. (2001) Circulation 103: 1194-1197Torzewski M et al. (2000) Arterioscler Thromb Vasc Biol 20: 2094-2099Koenig W et al. (1999) Circulation 99: 237-242Ridker P et al. (1997) New Engl J Med 336:973-979Mendall M et al. (1996) BMJ 349:462-466Rifai N et al. (2001) Clin Chem 47 : 403-411


35

Reference Values CRP

Serum / Plasma: < 0.068 - 8.2 mg/l

Stool: < 56 ng/ml

Umbilical cord blood: < 0.6 mg/l

Urine: < 6 ng/l

Sandwich ELISA technology that uses a pair of highly specific an-tibodies which provides high sensitivity (4 ng/ml) Assay designed to be suitable for processing small amounts of specimen also

also available as 1-point-calibration test (Cat. No. K 9720s

Matrix Serum, Plasma, Urine, Stool, dried blood

Sample volume 10 µl (Ser.; Pl.) 50 µl (Urine) 15 mg (Stool) 50 µl (dried blood)

Test principle ELISA Cat. No. K 9710s

CRP

IDK® PMN ElastasePMN Elastase from human polymorpho-nuclear granulocytes is a glycoprotein of 30 kDa and belongs to the family of serine proteases. The release of active PMN Elastase occurs after the challenge of the azurophilic granules of neutrophil granulocytes or during the degradation of these cells. The determination of PMN Elastase in plasma is used for recording inflammatory reactions involving neutrophil granulocytes. PMN Elastase strengthens the MPO-H2O2 system. Clinically silent inflammation on genital tract organs can be diagnosed by measuring PMN Elastase in semen.

Indications • Bacterial infection, sepsis • Adult Respiratory Distress Syndrome (ARDS) • Subclinical genital tract infection or inflammation

ReferencesEggert-Kruse W et al. (2008) Int J Androl. 2008 Jan 10. [Epub ahead of print] Langhorst J et al. (2008) Am J Gastroenterol 103(1):162-9Langhorst J et al. (2007) Z Gastroenterol 45: P261Langhorst J et al. (2005) Inflamm Bowel Dis 11(12):1085-91Silberer H et al.(2005) Clin Lab 51(3-4):117-26Heinichen et al. (1995) Clin Lab 41: 539-545Eggert-Kruse et al. (1996) Fertility and Sterility 65 : 1202-1209Oremek et al. (1995) MTA 10, 273-278.Windolf I et al. (1994) Unfallchirugie 20:239-250 Wolff H et al. (1991) J Andrology 12:331-34


36


sample also

Sandwich ELISA technology with a highly specific pair of

antibodies

Reference Values PMN Elastase

Stool < 62 ng/ml

Serum 19 - 78 ng/ml

also available as 1-point-calibration test

(Cat. No. K 6830)

IDK® PMN Elastase

Matrix Stool

Sample volume 15 mgTest principle ELISA Cat. No. K 6840

IDK® PMN Elastase

Matrix Serum, Plasma, Seminal Plasma

Sample volume 20 µlTest prinicple ELISA Cat. No. K 6841

also available as 1-point-calibration test

(Cat. No. K 6831)

RelaxinRelaxin has a molecular weight of 6500 Da and belongs to the Insulin peptide family. The main function of this peptide is the relaxing effect on smooth muscule. Because of the increased concentration of relaxin during ovulation and pregnancy, it is very well known to physicians in the field of gynaecology and fertility. Besides its rela-xing function on the cervix, uteri and os uteri during delivery, relaxin can also act vasoactivly on the vasculatory system and thereby influences the hemodynamic system.

Receptors for relaxin have been described in the brain (interaction with ADH secretion) and in the heart muscle (influence on the heartbeat rhythym). Recent studies have shown a correlation between relaxin and oxidative stress. Bani et al. provide evidence that the addition of relaxin to the reperfusion solution after ischaemia protects the myocardial tissue in rat hearts against oxidative changes and stress. They studied the production of malondialdeyde (metabolite of lipid peroxida-tion) and myeloperoxidase (marker for inflamatory processes produced by neutro-phils). Both markers are clearly decreased in comparison to controls. Consequently the damage of the myocardial tissue and mortality are significantly decreased.

Indications • Protection from reperfusion/ischaemia

ReferencesBoehnert MU et al. (2008) Transplant Proc 40(4):978-80Schöndorf T et al. (2007) Gynecol Endocrinol 23(6):356-60Schöndorf T et al. (2007) Clin Lab 53:193-198Dschietzig et al. (2004) Abstract of Fourth Intern Conference on Relaxin & Related Peptides, Jackson Hole, USAHeringlake M et al. (2004) J Appl Physiol 97(1):173-79 Hocher B et al. (2004) Circulation 109: 2266-2268Nistri S et al. (2003) FASEB J 17 (14) 2109-2111Arnoldt C et al. (2002) Orthopedics 25:669-73Dschietzig R, Stangl K (2002) CMLS 59: 1-13 (Review)Armbruster FP et al. (2001) Eur J Med Res 6:1-9Armbruster et al. (2001) in Relaxin 2000; Proceed third Intern Conference on Relaxin & Relates Peptides, 2-27 October 2000, Broome, Australia , 273-274. eds. Tregear GW, Ivell R, Bathgate RA,WAde JD. Kluwer Academic Publishers. ISBN 1-4020-0068-5Bani D (1997) Gen. Pharmac. 28:13-22Bani D et al. (1998) A. J. Patholoy 152:1367-1375


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Designed to be suitable for proces-sing small amounts of specimen Sandwich ELISA technology with a highly specific pair of antibodies

Cross reactivityNo cross reactivity was observed with:• Insulin• Zinc Insulin• Prolactin• Inhibin• Prorelaxin • Porcine relaxin

Sample volume 100 µlMatrix Serum, Tissue, Plasma, Urine Test principle ELISA Cat. No. K 9210

Relaxin (ELISA)

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Thiol-Status (Sulfhydryl status test)

Oxidative stress, or the production of oxygen-centered free radicals, has been hypothe-sized as the major source of DNA damage that in turn can lead to altered genetic expres-sion, disease, and aging of humans.

Serum protein thiol levels in blood are a direct measure of the in vivo reduction/oxi-dation (redox) status in humans, because thiols react readily with oxygen-containing free radicals to form disulfides. Moreover, serum thiols also reflect DNA repair capacity and the possible eventual accumulation of genetic damage, since a key DNA repair en-zyme, poly ADP-ribose polymerase (PARP), is thiol/disulfide redox regulated.

Serum protein thiols can possibly be used to estimate individual aging status. Data from Banne et al. (2003) strongly confirm an important role of oxidative stress in human disease development, and identify serum thiol status as a potential biochemical end-point useful in the assessment of aging.

ReferencesBanne AF et al. (2003) Anti Aging Med 6: 327-34Belch JJ et al. (1991) Br Heart J May; 65(5):245-248Dabrowski A, Gabryelewicz A (1992) Int J Pancreatol Dec; 12(3): 193-199 Collier A et al. (1979) Diabet Med 7(1): 27-30Ellmann G, Lysko H (1979) Anal Biochem; Feb; 93(1): 98-102Hu-ML: Methods-Enzymol. 1994; 233: 380-385Jocelyn PC (1987) Methods Enzymol ; 143: 44-67Langley SC et al. (1993) Pediatr Res; Mar; 33(3): 247-250Miesel R, Zuber M (1993) Inflammation. Oct; 17(5): 551-561Nguyen TT et al. (1993) J Burn Care Rehabil; Nov-Dec; 14(6): 602-609Riddles PW et al. (1983) Methods Enzymol 91: 49-60 Sitton NG (1986) Wright-V: Rheumatol Int 6(6): 251-254


Thiol Status (Sulfhydryl status test) Sample volume 20 µlMatrix Serum, Plasma, Urine, SynoviaTest principle colorimetricCat. No. K 1800

Simple methodology

Short incubation (30 min)

Requires only 20 µl as a sample volume

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Brochure Oxidative Stress March 2019 · 3 Oxidative stress – underlying cause of many diseases Oxidative stress represents an imbalance between the systemic manifestation of reactive