oxidant/antioxidant status in alopecia areata
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Oxidant/antioxidant status in alopecia areata

Antioxidants are substances that inhibit oxidation or inhibit reactions promoted by oxygen and peroxides. The chemical pathway of antioxidants is a highly regulated one. During inflammatory disorders, cells generate reactive oxygen species [ROS]. When produced in excess, ROS brings about an imbalance in the antioxidant pathway. This imbalance of pro-oxidants and antioxidants in which pro oxidants exceed in concentration is referred to as “oxidative stress”. Oxidative stress is very harmful to cells including cells in hair follicles.

When the amount of ROS increases in the cell, it reacts with the cellular constituents, particularly the fatty acids on the cell membrane and other molecules such as carbohydrates, lipids, proteins and nucleic acids. One reaction leads to another and the cell goes awry in its normal functioning causing cell injury and finally cell death.

Since alopecia areata is considered an autoimmune disease mediated by T lymphocytes, an analysis of the role played by lipid peroxidation and antioxidant enzymes in patients with alopecia areata was conducted. The study involved the evaluation of malon-di-aldehyde [MDA] and nitric oxide [NO] and testing of activities of superoxide dismutase [SOD] and xanthine oxide [XO] in 24 Caucasian patients with alopecia areata and 20 controls matched according to the sex and age of patients.

Alopecia areata can be of three basic types:- patchy alopecia areata, Alopecia areata totalis (total scalp hair loss)and alopecia areata universalis (total sclap and body hair loss). of these, patchy alopecia areata is the most frequently occurring presentation. Hence the selected patients were confined to parameters such as patchy alopecia areata, non-alcoholism (alcohol significantly interferes with the antioxidant chemical pathway) and without any other autoimmune disease. Blood was taken from all the participating members when they were fasting and stored in -40 deg C for biochemical assays.

1. Assay to determine malon-di-aldehyde [MDA] in a given serum sample –
a. 1 volume of blood sample was mixed with 2.5 volumes of 10% [weight/volume] trichloroacetic acid to precipitate all the proteins.
b. The above sample was centrifuged to pellet the precipitated proteins and the supernatant (remaining fluid) retained.
c. To the supernatant, 0.67% thiobarbituricacid [TBA] was added and the mixture was heated in a boiling water bath.
d. The sample was cooled and the light absorption pattern was read using a light source with a specific wavelength of 532nm.
e. The values were compared with that of a standard solution of 1,1,3,3 tetramethoxypropane.
f. The results were expressed as nanomoles/milliliter [nmol/ml].
This method of MDA determination is known as the Draper and Harley method. It is based on the reaction of MDA with TBA at a temperature of 95 deg C, pH 2-3 for 15 minutes. The resultant reaction is the formation of a pink pigment, which is studied at an absorption peak of 532nm.

2. Superoxide dismutase [SOD] activity in a given sample –
This assay is based on the method of Sun et al. 1ml of the sample was mixed with 1ml of ethanol/chloroform mixture [5/3, volume/volume] and centrifuged. The clear top layer was dialyzed with phosphate buffer and xanthene oxidase was added as a superoxide generator. Nitroblue tetrazolium [NBT] was the indicator whose reduction is inhibited by xanthine oxidase. When the enzymes in the sample solution exhibit 50% inhibition of NBT reduction, this 50% inhibition point is defined as 1 unit and the SOD activity is expressed as unit(s)/ml [U/ml].

3. Xanthine assay –
This is the measure of uric acid formed from xanthine in the sample at pH 7.5 and temperature 35 deg C. By studying the samples at 293nm using a spectrophotometer, xanthine oxidase activity can be reported as U/ml which is the amount of 1micromole uric acid formed per minute.

4. Nitric oxide determination –
In the blood samples, nitric oxide (NO) degenerates very quickly to nitrate and nitrite. This degeneration is used as an indicator to measure the amount of NO present in the sample by indirectly measuring the amount of nitrates and nitrites based on the principle of the Greiss reaction. For the sample to be assayed it must first be deproteinized. Griess reaction does not help measure nitrates but only indicates its presence. Hence the total nitrate level is measured by adding copperized cadmium granules to the samples. This helps find the amount of nitrates converted to nitrites and can be studied using a spectrophotometer at 545 nm. A standard curve using serial dilutions of Sodium nitrate helps calculate the concentration of nitrates in the sample and is expressed as nanomoles/liter.

The results from all these assays were analyzed statistically using a Mann - Whitney test. The results indicated high levels of MDA, NO and enzymatic activity of XO and low SOD activity in the serum of alopecia areata patients as compared with their controls.

Logic behind these experiments –
The polyunsaturated fatty acids of phospholipids in the cell membrane are very important components required for normal functioning of mammalian cells. When this is destroyed the cells develop oxidative stress. MDA which is an end product of the oxidative stress is an indicating factor for the level and extent of lipid peroxidation.

Earlier studies had reported an increase in levels of TBARS [thiobarbituricacid reactive substances] in the plasma and erythrocytes and scalp tissues of alopecia areata patients especially during the onset of the disease. Also the amount of glutathione peroxidase [GSH-Px] which is an antioxidant enzyme like SOD that protects cells from damage caused by peroxided has been found to be in lower levels just as SOD in alopecia areata patients. A correlation can be drawn here with respect to antioxidants and MDA levels. A decrease in SOD activity may be responsible for increase in MDA levels.

In patients with inflammatory disorders, it has been shown that xanthine oxide [XO] which is present in normal tissues as xanthine dehydrogenase [XD] gets converted to XO. XO is an enzyme that catalyses the conversion of hypoxanthine to xanthine and xanthine to uric acid. So an increase in XO may be contributory factor to the oxidative stress in Alopecia areata patients.

Another phenomenon observed in inflammatory disorders and immune response is the generation of NO which is considered as a pro-inflammatory agent. When cytokines such as Tumor necrosis factor-alpha, Interferon-gamma, Interleukin-1 beta are stimulated during inflammation, NO is generated in a large scale. This reacts with the anions of superoxides to form a toxic component peroxynitrite. This is highly toxic and drives the cell to death.

Since the amounts of MDA, SOD, XO and NO in cells destroy antioxidant defense mechanisms, their levels in serum may be used as another yardstick for developing appropriate diagnostics not only for immune disorders but also for alopecia areata. The increased lipid peroxidation in AA may be related to an increase in NO level and XO activity and a decrease in SOD activity. These results suggest that lipid peroxidation and alterations in the oxidant-antioxidant enzymatic system may play a role in the pathogenesis of AA.

Oxidant/antioxidant status in alopecia areata references

  • Koca R, Armutcu F, Altinyazar C, Gurel A. Evaluation of lipid peroxidation, oxidant/antioxidant status, and serum nitric oxide levels in alopecia areata. Med Sci Monit. 2005 Jun;11(6):CR296-299. PMID: 15917721
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