Oxidative Stress Measurement

Oxidative stress is "a disturbance in the prooxidant/antioxidant balance and in favor of the former, leading to potential damage." Reactive oxygen species (ROS) are produced by both endogenous and exogenous processes. Eukaryotic organisms have developed a complex antioxidant network to counteract ROS to reduce or prevent the deleterious actions of ROS. Oxidative stress occurs when the generation of ROS in a system exceeds the system's antioxidant capability to neutralize and eliminate them. The imbalance can result from a lack of antioxidant capacity caused by disturbance in production, distribution, or by an overabundance of ROS from an environmental or behavioral stressor. If not regulated properly, the excess ROS can damage a cell's lipids, protein, or DNA, inhibiting normal function. Oxidative stress has been implicated in a growing list of human diseases such as cancer, Parkinson's disease, Alzheimer's disease, and aging process. One of the greatest needs in this field of research has been to assess the oxidative stress status in the biological systems. A variety of methods for the measurement of oxidative stress have been proposed. In the ITPC Lab, we have successfully established a number of measurements for oxidative stress/damage in a variety of biological samples with excellent accuracy and reproducibly:

Lipid peroxidation

Measurement of Malondialdehyde using HPLC-UV detection
One of the deleterious consequences of oxidative damage is lipid peroxidation, which involves hydrogen abstraction from fatty acids by free radicals such as •OH and once initiated is a self-propagating process. Malondialdehyde (MDA), a highly reactive three carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism, is one of the most intensively investigated aldehydes formed during lipid peroxidation. MDA readily combines with several functional groups on molecules including proteins, lipoproteins, and DNA. MDA-modified proteins show altered physico-chemical behavior and antigenicity. Thus, MDA is not only a useful biomarker for oxidative stress, but also has a significant pathophysiological implication.
The most common method for MDA measurement is the colorimetric or fluorimetric determination of MDA or MDA-like materials by thiobarbiruric acid (TBA) acid assay. Unfortunately, the TBA assay is intrinsically non-specific for MDA and is reactive to other compounds that may be present in biological samples. Employment of high-performance liquid chromatography (HPLC) with UV detection has improved the selectivity. The use of derivative agent 2,4-dinitrophenylhydrazine (DNPH), which reacts with MDA at low pH levels to form DNPH derivative with strong UV absorption, has greatly improve the sensitivity of this method. In our lab, we established this technique and have successfully used it to quantify MDA in plasma, serum, cultured cells, liver and brain tissues. This method can measure both free and total MDA, which includes free and protein-bound MDA.

Sample requirements and preparation:

Tissue samples
- Amount of sample: A minimum of 300mg, preferably 500mg tissue is needed for each assay
- Sample Preparation:
  - Snap freeze tissue samples in liquid nitrogen immediately after collection
  - Store snap-frozen tissues at -80ºC
  - Transport tissue samples to our Center CORE Lab on dry ice.
Plasma samples
- Amount of sample: A minimum of 200µl, preferably 500µl is needed for each assay.
- Sample Preparation:
  - Collect blood samples in EDTA containing tube
  - Separate plasma by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice.
C. Serum samples
- Amount of sample: A minimum of 200µl, preferably 500µl is needed for each assay.
- Sample Preparation:
  - Collect blood samples without using anticoagulant
  - Separate by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice.

Oxidative DNA Damage

Measurement of 8-hydroxy-2'-deoxyguanosine

Reactive species of oxygen induces a variety of forms of DNA damage, including base modifications, base-free sites, strand breakage, etc. The most prevalent product of DNA oxidation that is detected in genomic DNA in mammalian cells is 8-hydroxy-2'-deoxyguanosine (8-OHdG). If DNA is replicated prior to repair of the modified base, 8-OHdG can cause G to T transversion mutations. 8-OHdG has been demonstrated to be a stable biomarker for DNA oxidation.
HPLC with electrochemical detection is so far the most common method to determination of 8-OHdG. One of the major concerns of 8-OHdG analysis is the artifact during the process. Studies show that 8-OHdG levels differ depending on the DNA isolation method. Phenol and chloroform are known to enhance the 8-OHdG level in DNA. In our lab, we adapted an improved genomic DNA extraction method using chaotropic agent sodium iodide. This technique has been proven causing much less artifact than the phenol/chloroform method. We have applied this technique to measure the 8-OHdG level in cultured cells, liver and brains tissue with great sensitivity and reproducibility. Briefly, cells or tissue homogenates are lysized by lysis buffer containing Triton X-100 to obtain nuclei. Nuclei are then incubated with Proteinase K to release DNA from nucleus. Sodium iodide is added followed by isopropanol to selectively precipitate DNA. DNA then is digested by nuclease P1 and alkaline phosphatase to release nucleotides. Nucleotides are separated on a C18 reverse HPLC column, and 8-OHdG is detected by an electrochemical detector and 2-deoxyguanosine (2-dG) is detected by a UV detector. The ratio of 8-OHdG to 2-dG is calculated to assess the DNA oxidation in samples.

Sample requirements and preparation:

Tissue samples
- Amount of sample: A minimum of 300mg, preferably 500mg tissue is needed for each assay
- Sample Preparation
  - Snap freeze tissue samples in liquid nitrogen immediately after collection
  - Store snap-frozen tissues at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice.
Cell culture
- Amount of sample: A minimum 5 million, preferably 10 million cells is needed for each assay.
- Sample preparation
  - Scrap the cells off culture dish in 1 ml cold PBS and stored at -80ºC, and
  - Immediately transport samples to Center CORE Lab on dry ice.

Hydroxyl free radical formation Determination

Hydroxyl radical (•OH) is the most reactive and toxic oxygen-derived free radicals. Due to its short half-life, it is very difficult to measure •OH directly. It has been demonstrated that salicylate, an effective •OH scavenger, can be used to trap •OH and results in the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Unlike other spin trap agents used in •OH detection, salicylate is not normally found in tissue. At least one of its product, 2,3-DHBA, does not occur endogenously and once formed, it cannot be further metabolized. With the high sensitivity of coulometric electrochemical detection, salicylate trapping has been successfully used in the measurement of •OH. In this method, salicylate is administered to experimental animals or cultured cells several hours before the end of the treatments. Samples then are collected. Tissue homogenates, cell lysates, serum, or plasma are deproteinized with perchloric acid and the deproteinized samples can be injected directly to HPLC for analysis. The separation of salicylate adducts is perform on a C18 reversed column and detection is performed on a coulometric electrochemical detector. In our lab, we have applied this technique to measure the formation of •OH in rat brain tissue, liver tissue, plasma, and cultured cells.

Sample requirements and preparation:

Tissue samples
- Amount of sample: A minimum of 300mg, preferably 500mg tissue is needed for each assay
- Sample Preparation:
  - Snap freeze tissue samples in liquid nitrogen immediately after collection
  - Store snap-frozen tissues at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice.
Plasma samples
- Amount of sample: A minimum of 200µl, preferably 500µl is needed for each assay
- Sample Preparation:
  - Collect blood samples in EDTA containing tube
  - Separate plasma by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to our lab on dry ice.
Serum samples
- Amount of sample: A minimum of 200µl, preferably 500µl is needed for each assay.
- Sample Preparation:
  - Collect blood samples without using anticoagulant
  - Separate by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice.

Glutathione: Reduced and oxidized glutathione using HPLC-EC detection

To counter the continuous challenge by oxidative stress, cells have developed a complicated antioxidant system. Reduced glutathione (GSH) is the major non-enzymatic antioxidant in the biological system. The oxidized glutathione (GSSG) is formed by enzymatic oxidation catalyzed by glutathione peroxidase. GSH level and the ratio of GSH to GSSG are valuable indicators of the oxidative stress status in the biological system.
Numerous methods have been developed to quantify GSH and GSSG. Because the lack of strong chromophores and fluorophores in the chemical structure of GSH and GSSG, most of these methods require derivatization to increase sensitivity, and thus increase the sample processing time, which may lead to autooxidation of GSH. Recently, a HPLC method with electrochemical detection has been developed to quantify GSH and GSSG simultaneously. The advantages of this method include very short sample preparation time, thus minimize the GSH autooxidation and much higher sensitivity than other methods. In this assay, plasma/serum, cultured cells and tissues are deproteinized with perchloric acid and then directly injected into HPLC system. The separation is performed on a C18 reverse column and the detection of GSH and GSSG is performed on a coulometric electrochemical detector. We have successfully used this technique in the quantification of GSH and GSSG in rat brain tissue, plasma and cell cultures.

Sample requirements and preparation:

Tissue samples
- Amount of sample: A minimum of 300mg, preferably 500mg tissue is needed for each assay
- Sample Preparation:
  - Snap freeze tissue samples in liquid nitrogen immediately after collection
  - Store snap-frozen tissues at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice
Plasma samples
- Amount of sample: A minimum of 200µl, preferably 500 µl is needed for each assay
- Sample Preparation:
  - Collect blood samples in EDTA containing tube
  - Separate plasma by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice
Serum samples
- Amount of sample: A minimum of 200µl, preferably 500 µl is needed for each assay
- Sample Preparation:
  - Collect blood samples without using anticoagulant
  - Separate by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice
Cell culture
- Amount of sample: A minimum 5 million, preferably 10 million cells is needed for each assay
- Sample preparation:
  - Scrap the cells off culture dish in 1 ml cold PBS and stored at -80ºC
  - Immediately transport samples to Center CORE Lab on dry ice

Measurement of total antioxidants capacity - TEAC assay

Reactive oxygen species (ROS) are produced by both endogenous and exogenous processes. Eukaryotic organisms have developed a complex antioxidant network to counteract ROS to reduce the deleterious actions of ROS. The Total Antioxidant Capacity (TAC), a parameter that provides information on the overall status of antioxidants within a biological sample, has proven to be a useful indicator for determining the ability of an organism to mitigate the potential damage produced by ROS. Various methods have been developed to assess TAC. The Trolox Equivalent Antioxidant Capacity (TEAC) assay, a method based on the scavenging of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical (ABTS•+), has been one of the most widely used methods. The original method used the methmyoglobulin radical to generate the ABTS•+ substrate. However, use of this method has been limited due to the length of time needed to perform this assay, lack of reproducibility, and expense. Recently, an improved technique of TEAC assay was developed using pre-formed ABTS•+. In our lab, we scaled this assay to a 96-well, high throughput format. The ABTS•+ is prepared by the reaction of ABTS with potassium persulfate 12-16 hours before use. Samples or Trolox standards are reacted with ABTS•+, and the absorbance is measured at 660 nm on a plate reader after 5 minutes for the determination of TEAC. This method has been used to evaluate the total antioxidant capacity of tissue homogenate, plasma/serum and cell lysates. It is also useful in the examination of antioxidant potential of chemicals.

Sample requirements and preparation:

Tissue samples
- Amount of sample: A minimum of 300mg, preferably 500mg tissue is needed for each assay
- Sample Preparation:
  - Snap freeze tissue samples in liquid nitrogen immediately after collection
  - Store snap-frozen tissues at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice
Plasma samples
- Amount of sample: A minimum of 100µl, preferably 200 µl is needed for each assay
- Sample Preparation:
  - Collect blood samples in EDTA containing tube
  - Separate plasma by centrifugation c) Store plasma samples at -80ºC
  - Transport tissue samples to our Center CORE Lab on dry ice
Serum samples
- Amount of sample: A minimum of 200µl, preferably 500mg µl is needed for each assay
- Sample Preparation:
  - Collect blood samples without using anticoagulant
  - Separate by centrifugation
  - Store plasma samples at -80ºC
  - Transport tissue samples to Center CORE Lab on dry ice.

Detection of Reactive Oxygen Species Using Carboxy-H2DCFDA

Dichlorodihyrofluorescein diacetate (H2DCFDA) is commonly used to detect the generation of reactive oxygen species. The carboxy-H2DCFDA, which has two negative charges at physiological pH, has a better permeability across cell membranes than H2DCFDA. After carboxy-H2DCFDA diffuses into cells, its acetate groups are cleaved by intracellular esterases and reacts with hydrogen peroxide to form DCF, which is a strong fluorescent molecular.

Sample requirement and preparation:

Cells should be cultured in a 6-96-well culture plate. At the end of treatment, cells should be at ~80% confluent. Bring the cells in culture dishes to the ITPC (MS, B-60) near the end of treatment.

Comet assay

Measurement of DNA damage

The single cell gel electrophoresis (Comet Assay) is one such state-of-the-art technique for quantitating DNA damage from in vivo and in vitro samples of eukaryotic cells. This technique is rapid, non-invasive, sensitive, visual, and inexpensive compared to conventional techniques and is a powerful tool to study factors modifying mutagenicity and carcinogenicity. It is the only technique that directly measures DNA damage in individual cells and as a result has rapidly gained importance in the fields of genetic toxicology and human biomonitoring. Comet Assay measures double strand breaks (DSBs), single strand breaks (SSBs), alkali labile sites, and DNA repair.
Sample requirement and preparation:

Cell culture: Cells should be cultured in a 35 mm culture dish or equivalent size of culture vesicle. Cells should be ~80% confluent at the treatment. Bring the cells in culture dishes to Center CORE Lab before 10:00 a.m.
Blood samples: Blood samples should be collected in EDTA containing tube and place on ice immediately after collection. Bring blood samples to Center CORE Lab on ice within one hour of collection. Alternatively, blood samples can be preserved in preservation media, step-wise frozen and stored at -80 ºC. We will provide preservation tubes containing preservation media. Transport samples to Center CORE Lab on dry ice.

Fpg-modified Comet assay

Determination of oxidative DNA damage

Single Cell Gel Electrophoresis (SCGE), or Comet assay, is a simple and sensitive method to assess DNA strand break. Formamidopyrimidine glycosylase (Fpg) is a DNA repair enzyme. Fpg catalyzes the excision of damaged DNA bases including 8-OHdG sites, and this property has been successfully incorporated into Comet assay to detect the oxidative DNA damage, mainly the 8-OHdG formation.
In our lab, we have successfully applied Fpg-modified Comet assay to examine the oxidative DNA damage in rat white blood cells and cultured cells. It is worth to point out that this technique is very useful in monitoring the oxidative DNA damage in peripheral white blood cells in human. It is simple and needs as little as 2 µl blood.

Sample requirement and preparation:

Cell culture: Cells should be cultured in a 35 mm culture dish or equivalent size of culture vesicle. Cells should be ~80% confluent at the treatment. Bring the cells in culture dishes to Center CORE Lab before 10:00 a.m.
Blood samples: Blood samples should be collected in an EDTA containing tube and placed on ice immediately after collection. Bring blood samples to Center CORE Lab on ice within one hour of collection. Alternatively, blood samples can be preserved in preservation media, step-wise frozen and stored at -80 ºC. We will provide preservation tubes containing preservation media. Transport samples to our lab on dry ice.