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
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
1. Assay to determine malon-di-aldehyde [MDA] in a given serum
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
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
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.