The ‘radioenzymatic assays’ have gained their abundant acceptance and recognition for the
assay of aminoglycoside antibiotics e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin,
tobramycin, doxorubicin, cephalosporins, cephamycins, thienamycin, lincomycin, clindamycin, erythromycin,
clarithromycin, azithromycin, oleandomycin, spramycins etc ; and chloramphenicol (or
Chloromycetine). Importantly, the radioenzymatic assays are exclusively based upon the fact that the
prevailing inherent microbial resistance to the said aminoglycoside antibiotics and chloramphenicol
is predominantly associated with the specific as well as the critical presence of certain highly specialized
enzymes* that particularly render the ‘antibiotics’ absolutely inactive via such biochemical means as :
acetylation, adenylation, and phosphorylation.
It has been duly proved and established that :
aminoglycoside antibiotics—are susceptible to prominent attack by these critical and
specific enzymes as :
Aminoglycoside acetyltransferases (AAC) ;
Aminoglycoside adenylyltransferases (AAD) ;
Aminoglycoside phosphotransferases (APH).
Chloramphenicol—is prone to predominant attack by the enzyme :
Chloramphenicol acetyl transferases (CAT).
Mechanism of Action : The mechanism of action of these enzymes viz, AAC, AAD, and APH
are not the same :
Acetyltransferases [i.e., AAC]—invariably attack the most susceptible amino moieties (–NH2),
and to accomplish this critical function may require acetyl coenzyme A (AcCoA).
Adenylyltransferases [i.e., AAD] and Phosphotransferases [i.e., APH]—these enzymes usually
attack the most susceptible hydroxyl moieties (–OH), and specifically requires adenosine
triphosphate [ATP] i.e., another nucleotide triphosphate.
Applications : As to date quite a few AAC and AAD enzymes have been judiciously employed
for the radioenzymatic assays.
Example : Both the enzyme and the suitable radiolabelled cofactor [1 – 14C]** acetyl coenzyme
A, or [2 – 3H]*** ATP are used frequently in order to specifically radiolabel the ‘drug substance’ under
investigation.
Method— The various steps involved in the assay are as follows :
(1) Enzymes are normally prepared by anyone of the following two techniques,
(a) Osmotic Shock i.e., by breaking the cells of an appropriate microbial culture by exposing
than to a change of strength of solution therby affording a definite perceptible alteration in
the ‘osmotic pressure’, and
(b) Ultrasonic Sound-waves i.e., by breaking the cells of a suitable bacterial culture by means
of the high-frequency ultrasonic sound waves.
Thus, the said two methods do break open the cells to a considerable extent, and no purification
is required at all.
(2) Radiolabelled drug substance is subsequently separated from the ensuing reaction mixture
soonafter the said reaction has attained completion duly. Thus, the exact quantum of the extracted
radioactivity is observed to be directly proportional to the exact quantum of the drug substance present
in the given sample.
Note : Separation of two types of antibiotics are accomplished duly as stated under :
(a) Aminoglycoside Antibiotics—by binding them suitably to phophocellulose paper, and
(b) Chloramphenicol—by making use of an organic solvent.
Calibration
In a particular situation when the reactants are adequately present in enough quantum, and the
prevailing reaction attains completion in due course, one may conveniently plot a graph of the counts
per minute (min–1) Vs concentration of calibrator, which is found to be linear, as illustrated
Non-Isotopic Modification
The calibration accomplished by using the radiolabelled drug essentially needs either a Geiger
Müller Counter or a Scintillation Counter, for
measuring the ensuing radio activity (in mC) of the radioactive
chemicals, which being an enormously expensive
equipment, and a skilled technician. Therefore,
in order to circumvent these glaring untoward serious
problems one may adopt a photometric variation
of the aminoglycoside acetyltransferases [AAC] assay meticulously. For this the sulphydry reagent
viz., 5, 5′-dithiobis (2-nitrobenzoic acid) is incorporated carefully into the on-going assay-system.
Thus, the said reagent specifically interacts with the corresponding coenzyme A (reduced form)
duly generated thereby producing a distinct yellow-coloured product that may be quantitatively assayed
by using a previously standardized UV-Visible Spectrophotometer.
(a) Reactions : The two reactions are as follows :
(b) Aminoglycoside + Acetyl CoA —→ Acetyl – Aminoglycoside + CoASH
CoASM + DTNB —→ Yellow Product