In an oxidative reaction, potassium permanganate reacts with the mycolic acid component of _M.tuberculosis_ and gives rise to a unique saponified pattern specific for _M.tuberculosis_. This brown orange pattern makes it possible to identify _M.tuberculosis_ in a typical laboratory sample. The method avoids the complexity of other methods with special regard to save time and costs

Mohsen khatami

English

Nature Precedings

"The Oxidative Reaction of Potassium 
Permanganate with Mycolic Acids 
Leads to a Unique Diagnostic Pattern 
for Mycobacterium Tuberculosis"
"By Seyed Mohsen Khatami"
Abstract
In an oxidative reaction, potassium permanganate 
reacts with mycolic acid component of M.tuberculosis 
and gives rise to a unique saponified pattern specific 
for M.tuberculosis.This brown orange pattern make
possible to identify M.tuberculosis in a typical
laboratory sample. The method avoids the complexity
of other methods with special regard to save time and 
costs.
Introduction
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), killed 
approximately two million people worldwide in 1997 and has infected 
approximately 32% of the world's population (Dye et al., 1999). The unique 
characteristics of M. tuberculosis with special regards to its pathogenic 
consequences depend on unique characteristics of cell wall skeleton.
The isolated cell wall contains three heteropolymeric subunits composed of 
peptidoglycan, arabinogalactan (a complex heteropolymer composed 
primarily of arabinose and galactose), and mycolic acids. The currently 
accepted model of cell wall structure relegates the peptidoglycan and 
arabinosgalactan polymers largely to providing a scaffold for attaching and 
orienting mycolic acids in a direction perpendicular to the plane of the cell 
surface1 (see Fig 2). 
Other than the normal barrier functions of a cell wall skeleton involved in 
maintaining a rigid cell shape and turgor pressure against the variable 
osmolarity of the surrounding environment, mycobacteria and related genera
possess many unique characteristics which are directly attributed to the 
presence of mycolic acids in their cell wall.2, 3   The more important 
characteristics conferred by this structure include properties such as 
resistance to chemical injury, low permeability to hydrophobic antibiotic
substances,4 resistance to dehydration, and the ability to persist and thrive 
within the hostile environment of the macrophage phaglysosome.5-7
Mycolic acids are very long chain Į-alkyl, ǃ-hydroxy fatty acids that are
unique to mycobacteria and are greater than 80 carbons in Mtb8.Mycolic acid
melts at 53-55.5°, has a molecular weight of 1316 and its formula indicates
C88H172O4 or C88H176O4.
1
The mycolate acids of the Mycobacteria are among the longest and most 
highly functionalized of the mycolata. They occur with a wide variety of 
functional groups, both polar and non-polar, which give rise to the
characteristics HPLC profiles. These modifications occur at two points in the 
mero chain, referred to as distal (closest to the ǔ-end of the chain) and 
proximal (closest to the ǃ-hydroxy acid) 1.
The most widespread of the classes of mycolic acids are the Į mycolates, 
which may contain double bonds or cyclopropanes in cis configurations or 
double bonds or cyclopropanes in trans configurations with an adjacent
methyl branch. Every mycobacterial species which has been examined to date
produces Į mycolates. The next most widely distributed class of mycolic acids 
are the ketomycolates. These are found in many different species; regardless 
of growth rate or status as pathogens or saprophytes.Methoxymycolates
appear, with five exceptions, to be present only in pathogenic species, and 
primarily in slow-growers. Interestingly, methoxymycolates in all cases appear 
to co-occur in species which also produce ketomycloate1.
Oxygenated mycolates such as ketomycolate and methoxymycolate are
extremely widespread and only a handful of species appear to be capable of 
maintaining cell wall function without the presence of such mycolates.
In spite of this fact there is very little actual evidence to suggest what 
physiological role such modifications play in the cell wall.
Ketomycolate is likely to be very active as a hydrogen-bond acceptor.
Methoxymycolate, which has only an ether linkage, is less likely to be very 
active in this regard. It is noteworthy that the combination of Į mycolate with 
methoxymycolate as the only oxygenated species is not a naturally-occurring 
combination, whereas the combination of only Į and ketomycolate does 
occur1.
M. tuberculosis produces three main types of mycolic acids: alpha-, methoxy-, 
and keto-. Alpha-mycolic acids comprise at least 70% of the mycolic acids 
present in the organism and contain several cyclopropane rings. Methoxy-
mycolic acids, which contain several methoxy groups, comprise between 10% 
and 15% of the mycolic acids in the organism. The remaining 10% to 15% of 
the mycolic acids are keto-mycolic acids, which contain several ketone groups. 
The presence of mycolic acids gives M. tuberculosis many characteristics that 
defy medical treatment. They lend the organism increased resistance to
chemical damage and dehydration, and prevent the effective activity of 
hydrophobic antibiotics (see Fig 2). In addition, the mycolic acids allow the 
bacterium to grow readily inside macrophages, effectively hiding it from the
host's immune system1,9. In pathogenic mycobacteria, but not in non-
pathogenic species, mycolic acids are modified with cyclopropyl groups at 
relatively conserved positions in the meromycolate chain (see Fig 1).
These cyclopropane residues are added to Mtb mycolic acids by a family of S-
Adenosy Methionine dependent methyltransferases that exhibit exquisite 
substrate specificity for their lipid substrates10,11. Although cyclopropanation 
of mycolic acids is observed only in pathogenic mycobacterium such as Tb
complex, little is known about the physiological function of this modification.
Potassium permanganate is highly reactive under normal chemical 
coundition.It will oxidize a wide variety of inorganic and organic substances.
2
Potassium permanganate (Mn 7+) is reduced to mangances dioxide (MnO2) 
(Mn 4+) which precipitates out of solution (Hazen and Sawer, 1992). All 
reactions are exothermic.
Under normal or alkaline condition, the half-reaction is (CRC, 1990):
Potassium permanganate oxidizes lipids at the site of double bonds and 
therefore can be used to determine the position of unsaturations in lipid
chains. According ALEX LESUKt AND R. J. ANDERSON "It is evident that 
oxidation of mycolic acid by permanganate was not sufficiently drastic to 
break down the large mycolic acid molecule into smaller recognizable units" 
but they emphasize "the principal product that was recovered differed but 
slightly in melting point and equivalent weight from the original acid. A small
quantity of acids which had an equivalent weight of 741 and melted at 61- 68” 
was isolated but the material was non-crystalline and was apparently a
mixture 12". The oxidizing function of potassium permanganate leads to its
reduction, creating an alkaline condition proper enough for creation of a
saponification of fatty acid, lipid and ester. Saponification is the hydrolysis of 
an ester under basic conditions to form an alcohol and the salt of a carboxylic
acid. Saponification is commonly used to refer to the reaction of a metallic 
alkali (base) with a fat or oil to form soap. Saponifiable substances are those 
that can be converted into soap. The oxidation of mycolic acids by potassium 
permanganate create such condition that lead to occurrence of a 
saponification process. we, in this experiment will use this characteristic as a
prefect method in order to easily, rapidly and cheaply identify M.tuberculosis 
in clinical samples compared with other methods.
Material and methods
We used in our experiment of very strongly positive sample confirmed by
PCR, culture and smear versus a negative sample confirmed by PCR, culture 
and smear. The used potassium permanganate was of high concentration. In 
the following we explain the procedures we developed for the objective of the 
experiment.
1-One milliliter of both positive and negative samples of type bronchial 
specimen were centrifuged at 12000 rpm for 5 minutes.
2-The supernatants were discarded.
3-The pellets were suspended in 500 micro liter mucolysin and processed by 
shaking under 5 minutes at room temperture.
4-The contents of tubes were centrifuged again at 12000 rpm for 5 minutes.
5-The supernatants were discarded and the pellets were washed two times by
500 micro liter sterile water as the above-mentioned procedures in order to 
washing away the remaining of mucolysin and finally the pellets suspended in
max 15 micro liter sterile water.
6-10 micro liters of suspensions were placed on two opposite and adjacent 
points of a microscopic slide and incubated at 56 °C under 6 minutes in order
3
to fix the mycobacterium tuberculosis in positive sample on microscopic slide.
7-When the slide was dehydrated, it was transferred to distillated water for 
two seconds and the slide again dried at room temperture.
8-When the slide was dehydrated at room temperature, we transferred 5
micro liters sterile water on both dehydrated points and added 10  micro liters 
highly concentrated potassium permanganate under mixing condition. The 
slide incubated again at 56°C until the slide was dehydrated.
9-The dehydrated points on slide washed by sterile water until the excess of 
color of potassium permanganate removed.
10-After drying the slide at r00m temperature the slide subjected for
microscopic investigation.
Result:
The microscopic investigation provided a distinguishing difference between
the positive sample and negative sample. The positive sample characterized by 
lightly deep to deep brown pattern indicating the consequences of oxidation of 
mycolic acids from M.tuberculosis by potassium permanganate described
above. Such pattern was not observed in negative sample (Fig 3).
Discussion
The results of our experiment indicate that the oxidation of  mycolic acids
from M.tuberculosis by potassium permanganate inspire a perspective for 
emergence of a relatively easy, time-saving and cheap method intending the
identification of M.tuberculosis compared with other methods including PCR
and Ziehl-Neelsen staining and etc . A statistically raw analysis of more than
hundred samples indicates that the specificity of this method is less than 50%
compared with the golden standard but the sensitivity of this method is very
high. The logic interpretation of this analysis is that not necessarily all positive
indicated samples by this method can be confirmed by culture, PCR or other
method but all negative indicated samples can be confirmed by culture, PCR 
and other methods. . We are statistically engaged to determine the magnitude 
of accuracy the method can provide.
I express my gratitude to the head of Baqiyatallah Clinical Laboratory
and other kind colleagues for the fine cooperation during the 
performance of this work. Relating every question in connection 
with this work can correspondence be done through the e-mail 
address: mohsen_khatami3@yahoo.com
References
1. Barry et al. (1998). "Mycolic acids: structure, biosynthesis, and 
physiological   functions." Prog. Lipid Res. 37(3): 143-79. 
2. Minnikin, D. E., in The Biology of the Mycobacteria, Vol, eds C. Ratledge
and J. Standford. Academic Press, London, 1982, pp. 95-184.
3. Rastogi, N., Res. Microbial., 1991, 142, 464-476.
4
4. Nikaido, H., Science, 1994, 264, 382-388.
5. Barry, C. E., III and Mdluli., Trends Microbiol., 1996, 4, 275-281.
6. Clemens, D. L., Trends Microbiol., 1997, 5, 383-385.
7. Armstrong, J. A. and D'Arcy Hart, P., J. Exp. Med., 1971, 134, 713-740
 8. Barry, C. E., 3rd, Lee, R. E., Mdluli, K., Sampson, A. E., Schroeder, B. G.,
9. Takayama, Wang, and Besra (2005). "Pathway to synthesis and processing 
of mycolic acids in Mycobacterium tuberculosis." Clin. Microbiol. Rev. 18(1):
81-101.
10.  Barry, C. E., 3rd, Lee, R. E., Mdluli, K., Sampson, A. E., Schroeder, B. G.,
11. Schroeder, B. G., and Barry, C. E., 3rd. (2001) Bioorg Chem 29, 164-177 
12. ALEX LESUKt AND R. J. ANDERSON ‘THE CHEMISTRY OF THE
LIPIDS OF TUBERCLE BACILLI’(From the Department of Chemistry, Yale 
University, New Haven), (Received for publication, July 26, 1940 in the
journal of biological chemistry).
Figure legends
Figure 1: An example of a A) Į-mycolic acid, B) Methoxy mycolates, C) 
Ketomycolates from Mycobacterium tuberculosis
5
Figure 2: Schematic diagram of the cell envelope of M.tuberculosis.Exteroir to 
the cell membrane (CM)  and peptidoglycan (PG), mycolic acids are covalently
linked to the arabinogalactan (AG), forming a thick hydrophobic layer. 
Mycolic acids in the form of trehalose dimycolate (TDM) are noncovalently
associated with the cell envelope. 
6
A                      B 
C                      D
E                     F
G                    H
    I                  J
Figure 3: A, B, C, D, E, F, G, H, I and J indicating positive samples. 
7
A           B
Figure 4: A and B indicating negative samples.
8


in the journal of biological chemistry).

Mycolic acids: structure, biosynthesis, and physiological functions.&quot; Prog. Lipid Res.

Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis.&quot;

The Oxidative Reaction of Potassium Permanganate with Mycolic Acids Leads to a Unique Diagnostic Pattern for Mycobacterium Tuberculosis

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The Oxidative Reaction of Potassium Permanganate with Mycolic Acids Leads to a Unique Diagnostic Pattern for Mycobacterium Tuberculosis

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