Test metodları

ELISA:

This assay is based on the sandwich ELISA technique. The wells of the provided microplate are pre‑coated with an antibody that specifically recognizes (human,rat etc.) (name of parameter). Standards and samples are introduced into the wells, where the target antigen binds to the immobilized antibody. Following this step, a biotin‑labeled detection antibody specific to (human,rat etc.) (name of parameter)is added, forming an antibody–antigen–antibody complex. Subsequently, Avidin conjugated with Horseradish Peroxidase (HRP) is introduced and binds to the biotinylated antibody. After incubation, any unbound substances are removed through washing. A substrate solution is then added to each well. In the presence of the HRP enzyme, a colorimetric reaction occurs, producing a blue color. The reaction is stopped by adding a stop solution, which changes the color from blue to yellow. The absorbance is measured at 450 nm (±2 nm) using a spectrophotometer. The intensity of the color is directly proportional to the concentration of (human,rat etc.) (name of parameter)in the sample. The concentration of the analyte can be determined by comparing the sample absorbance values to a standard calibration curve.

COLORIMETRIC:

TOTAL ANTIOXDANT STATUS (TAS) (mmol/L)

TAS levels were measured using commercially available kits (Relassay, Turkey). The novel

automated method is based on the bleaching of characteristic color of a more stable ABTS

(2,2 ′ ‑ Azino‑bis(3‑ethylbenzothiazoline‑6‑sulfonic acid)) radical cation by antioxidants. The

assay has excellent precision values, which are lower than 3%. The results were expressed as

mmol Trolox equivalent/L (Erel O. A novel automated direct measurement method for total

antioxidant capacity using a new generation, more stable ABTS radicalcation. Clin Biochem

2004;37:277‑85.)

(Relassay,Turkey)

Erel O. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin Biochem. 2004 Feb;37(2):112‑

TOTAL OXIDANT STATUS (TOS) (µmol/L)

TOS levels were measured using commercially available kits (Relassay, Turkey. In the new

method, oxidants present in the sample oxidized the ferrous ion‑o‑dianisidine complex to

ferric ion. The oxidation reaction was enhanced by glycerol molecules abundantly present in

the reaction medium. The ferric ion produced a colored complex with xylenol orange in an

acidic medium. The color intensity, which could be measured spectrophotometrically, was

related to the total amount of oxidant molecules present in the sample. The assay was

calibrated with hydrogen peroxide and the results were expressed in terms of

micromolar hydrogen peroxide equivalent per liter (μmol H2O2 equivalent/L). ( Erel O. A

new automated colorimetric method for measuringtotal oxidant status. Clin Biochem

2005;38:1103‑11. ).

(Relassay,Turkey)

Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005 Dec;38(12):1103‑11.

OXIDATIVE STRESS INDEX (OSI)

The ratio of TOS to TAS was accepted as the oxidative stress index (OSI). For calculation, the

resulting unit of TAS was converted to μmol/L, and the OSI value was calculated according to

the following Formula : OSI (arbitrary unit) =

TOS (μmol H2O2 equivalent/L) / TAC (μmol Trolox equivalent/L). (1‑3).

1. Yumru M, Savas HA, Kalenderoglu A, Bulut M, Celik H, Erel O. Oxidative imbalance in

bipolar disorder subtypes: a comparative study. Prog Neuropsychopharmacol Biol Psychiatry.

2009 Aug 31;33(6):1070‑4.

2. Kosecik M, Erel O, Sevinc E, Selek S. Increased oxidative stress in children exposed to

passive smoking. Int J Cardiol 2005;100:61–4.

3. (Harma M, Harma M, Erel O (2003) Increased oxidative stress in patients with

hydatidiform mole. Swiss Med Wkly 133:563‑536).

Catalase (CAT) U/L

This colorimetric assay involves two steps. Sample is first incubated with a known amount of

hydrogen peroxide. Sample converts hydrogen peroxide to water and oxygen. The ratio is

proportional to the concentration of catalase. The enzyme is stopped and the remaining

hydrogen peroxide, following a fixed incubation period, is determined using a chromogen.

The resulting absorbance is measured at 405 nm and the obtained results are expressed as U/L.

(Relassay, Turkey)

Góth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta. 1991 Feb 15;196(2‑3):143‑151.

Thiol/Disulfide Homeostasis (µmol/L)

Tests were measured using a novel automatic and spectrophotometric method developed by Erel and Neselioglu*

which is avaliable commercially (Rel Assay Diagnostics, Turkey) In this method, dynamic and reducible disulfide bonds

in the samples were reduced to free functional thiol groups by using sodium borohydride. In order to prevent the reduction

of unused reduced sodium borohydride to dithionite‑2 nitrobenzoic (DTNB), NaBH4 was removed with formaldehyde. Native thiol (NT) and total thiol (TT)

levels were determined after reaction with DTNB and their levels were measured ultimately. Half of the difference of the result obtained

by the subtraction of native thiol amount from total thiol content indicated the disulfide (DS) level.

(Relassay, Turkey)

Erel O, Neselioglu S. A novel and automated assay for thiol/disulphide homeostasis. Clin Biochem. 2014 Dec;47(18):326‑32. doi: 10.1016/j.clinbiochem.2014.09.026. Epub 2014 Oct 7. PubMed PMID: 25304913.

Paraoxonase‑1 PON‑1) U/L

Measurement of paraoxonase activity;

Paraoxonase activity was measured using

commercially available kits (Relassay, Turkey).

The rate of paraoxon hydrolysis (diethylpnitrophenylphosphate)

was measured by monitoring the increase of

absorption at 412 nm at 37 °C. The amount of generated p‑nitrophenol

was calculated from the molar absorption coefficient at pH 8.5, which

was 18.290 M−1 cm−1 Paraoxonase activity was expressed as U/L serum

(Relassay, Turkey)

Myeloperoxidase (MPO) U/L

MPO posseses various catalytical activities. It exhibits the main catalytical activity by the

production of hypochlorous acid (HClO) from hydrogen peroxide (H2O2) and chloride anion,

Cl‑ (or halide). MPO also exhibits peroxidase activity that catalyzes oxidation of a number of

substrates by H2O2. These reactions categories have been widely used to assess the

activities of MPO.

The Relassay Myeloperoxidase Chlorination Activity Assay Kit and The Relassay

Myeloperoxidase Peroxidation Activity Assay Kit are quantitative and colorimetric assay kits

for measuring the myeloperoxidase activity within a sample. In the The Relassay

Myeloperoxidase Chlorination Activity Assay Kit, MPO catalyzes the formation of

hypochlorous acid, which reacts with taurine to form taurine chloroamine. Taurine chloroamine reacts with the chromophore TNB, resulting in the formation of the colorless

product DTNB. One unit of MPO activity is defined as the amount of enzyme that hydrolyzes

the substrate and generates taurine chloramine to consume 1.0 μmole of TNB per minute.In

the The Relassay Myeloperoxidase Peroxidation Activity Assay Kit, MPO catalyzes odianisidine

to colored o‑dianisidyl radical using H2O2. The increasing absorbance is

monitored at 412 nm and the activity is measured kinetically. This kit can be used manually

and easily adapted to automated analyzers.

(Relassay, Turkey)

1‑ Kusuma KS*, Vasudha KC, Vanitha Gowda MN. A Comparative Study of Serum Myeloperoxidase Activity in Type 2 Diabetes and Diabetic Nephropathy. Biomedical Research (2009) Volume 20, Issue 3: 198‑204

2‑ Krueger AJ, Yang JJ, Roy TA, Robbins DJ, Mackerer CR. An Automated Myeloperoxidase Assay. Clin Chem 1990;36:158

3‑ Worthington Enzyme Manual. Freehold, NJ: Worthington Biochemical Corp;1972.43‑55.

Zinc (ZN) μg/dl

Zinc found in the samples change the red‑orange color of 5‑Br‑PAPS to light pink under alkaline conditions.

The change of absorbance at 548 nm is proportional to total zinc level in the sample.

The assay can be calibrated with zinc sulfate dissolved in deionized water.

(Relassay, Turkey)

Copper (CU) μg/dl

Copper found in the samples change the red‑orange color of DiBr‑PAESA to violet under acidic conditions.

The change of absorbance at 572 nm is proportional to total copper concentration in the sample.

The assay can be calibrated with copper sulfate dissolved in deionized water.

(Relassay, Turkey)

Paraoxonase and Arylesterase activities;

Paraoxonase and arylesterase activities were measured using

commercially available kits (Relassay, Turkey).

The rate of paraoxon hydrolysis (diethylpnitrophenylphosphate)

was measured by monitoring the increase of

absorption at 412 nm at 37 °C. The amount of generated p‑nitrophenol

was calculated from the molar absorption coefficient at pH 8.5, which

was 18.290 M−1 cm−1 Paraoxonase activity was expressed as U/L

serum. Phenylacetate was used as a substrate to measure the

arylesterase activity. Enzymatic activity was calculated from the molar

absorption coefficient of the produced phenol, 1310 M−1 cm−1. One unit

of arylesterase activity was defined as 1 μmol phenol generated per

minute under the above conditions and expressed as U/L

(Relassay, Turkey)

Ischemia Modified Albumin (IMA)

The colorimetric assay format quantitatively measures

unbound cobalt remaining after cobalt‑albumin binding

has occurred. Thus, with reduced cobalt‑albumin binding,

there is more free, unbound cobalt detected, resulting

in elevated assay levels.

Glutathione Peroxidase (GPx) (U/L)

This method is based on that of Paglia and Valentine. Glutathione Peroxidase (GPx) catalses of the

oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione (GSSG) is

immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP. The decrease in absorbance at 340 nm is measured

Referanslar

Paglia, D.E. and Valentine, W.N., J. Lab. Clin. Med., 1967; 70: 158.

Prohaska, J.R., Oh, S.H., Hoekstra, W.G. & Ganther,

H.E. Biochem. & Biophys. Res. Comm. 1977; 74: 64.

Kraus, R.J. & Ganther, H. E. Biochem. & Biophys. Res. Comm 1980; 96: 1116.

Otto Scientific Cat.No. Otto2085

Malondialdehyde (MDA) nmol/L

The MDA level was determined by a method based

on the reaction with thiobarbituric acid (TBA) at 90–100_C

. In the TBA test reaction, MDA or MDA‑like

substances and TBA react with the production of a pink

pigment with a maximum absorption at 532 nm. The

reaction was performed at pH 2–3 at 90_C for 15 min. The

sample was mixed with two volumes of cold 10% (w/v)

trichloroacetic acid for the precipitation of protein. The

precipitate was pelleted by centrifugation, and an aliquot of

the supernatant was reacted with an equal volume of 0.67%

(w/v) TBA in a boiling water bath for 10 min. After

cooling, the absorbance was read at 532 nm.

Otto Scientific Cat.No. Otto1001

Micro Protein mg/dL

Bradford yöntem

Dokular 1:9 oranında (örnek 0.1 gr doku, 0.9ml tampon) fosfat tamponunda (50 mmol. lık pH:7.40 Fosfat Tamponu) homojenize edildi, 7000 rpm +4°C'de 5 dk. santrifüj edildi.

Nitric Oxide (NO) (µmol/L)

NO is easily oxidized to form N0² in vivo or in aqueous solution, and a reddish azo compoun is formed with the color developing agent, and the concentratıon of the azo compound is linearly related to the concentration of NO.

The concentration of NO can be calculated indirectly by measuring the OD value at 550 nm.

Protein Carbonyl nmol carbonyl/mg.

Protein carbonyl groups are a significant biomarker of oxidative stress. The DNPH tagging of protein carbonyls has become one of the most common methods for measuring oxidative stress.

The resulting DNP hydrazones from this reaction can be easily quantified at 375 nm.

Super Oxide Dismutase (SOD) U/ml

The role of superoxide dismutase (SOD) is to accelerate the reaction that converts the toxic superoxide radical (O2•), produced in oxidative energy processes, into hydrogen peroxide and molecular oxygen.

This method uses xanthine and xanthine oxidase (XOD) to generate superoxide radicals, and these radicals react with 2‑(4‑iodophenyl)‑3‑(4‑nitrophenyl)‑5‑phenyltetrazolium chloride (INT) to form a red formazan dye.

Superoxide dismutase activity is measured in a fully automated device using the reaction type endpoint 505 nm.

Total Glutathione and GSSG

Total GSH were determined by the method

described by Alisik et al. [M. Alisik, S. Neselioglu, O. Erel, A colorimetric method to measure oxidized,

reduced and total glutathione levels in erythrocytes, J. Lab. Med. 43 (5) (2019)

269–277]. GSH levels of supernatant samples were

measured using the Ellman method that was using 500 mM Tris solution

(pH: 8.2).