Absorption of Barbiturate Across A Biological MembraneEffect of pH and Lipid Solubility
Introduction :
Goldfish , the test animal being used in this experiment , have been
used for a number of years for determing the toxicity of a variety of drugs .
A number of investigations have established the fact that a reciprocal
relationship exists between the time of occurrence of a suitable pharmacologic
effect (time of death or overturn time ) and both the concentration of drug in
the aqueous medium to which the fish are exposed and the absorption rate of
the drug . Mathematically , this relationship is expressed by the following
equation , as proposed by Levy and co-workers .
1/T = ( K a / L ) C
(Eq.1)
Where T = the time required to elicit a particular pharmacologic response ( in
this experiment time of death ) , L = the amount of drug required in the fish to
elicit the pharmacologic response , C = the initial concentration of drug
present in the aqueous medium , and K a = the apparent , first-order absorption
rate constant , which includes membrane surface area variations will affect the
magnitude of the absorption rate constant , it is necessary to use the fish of
one size in any experiment . The assumptions on which this relatioship is
based have been discussed , in details , by levy and Gucinski (2).
It is the purpose of this experiment to demonstrate the influence of two
physiochemical properties (i.e., degree of ionization and lipid solubility) on
the rate of absorption of two barbiturates across the goldfish membrane .
Procedure:
Place a single fish into 100 ml of test solution contained in a 250 ml
capacity glass beaker . Determine the time of death (the time when gill and
mouth movement have ceased ) ,and subsequently record the weight of each
fish . Five fish are to be tested in each of the following solutions :
1.
Pentobarbital sodium , 30 mg % in pH 5.9 phosphate buffer (0.05 M )
2.
Pentobarbital sodium , 30 mg % in pH 6.9 phosphate buffer (0.05 M )
3.
Pentobarbital sodium , 30 mg % in pH 8.0 phosphate buffer (0.05 M )
4.
Secobarbital sodium , 30 mg % in pH 5.9 phosphate buffer (0.05 M )
Note : Adjust the phosphate buffer system given in U.S.P. XVII such that the
total salt concentration is 0.05 M . Measure the pH of the drug solutions
before and after the experiment .
Treatment of Data :
1. List the assumptions upon which Eq. 1 is based .
2. With the aid of experimental data from your classmates , determine the
mean T and 1/T , based on all the animals used in the experiment for
the section , for each of the barbiturate solutions .Also calculate the
standard error and deviation for each mean .
3. Using the student “t” test and the T values , determine whether there is
statistically significant difference between the means of drug
treatments (1) and (2) and (3) and (4) .
4. Plot the mean 1/T values versus pH . Physiochemically explain the
results obtained . The pKa of pentobarbital is 8.1 .
5. The values of L (Eq. 1) are 195 and 38.0 micrograms per gram of fish
for pentobarbital and secobarbital , respectively .Using the mean 1/T
values , calculate the Ka values for the four test barbiturate solutions
.NOTE : Be sure you use proper units for the parameters in Eq. 1 .
6. Determine whether there is a statistical difference between 1/T for
secoparbital (pKa = 7.9) and pentobarbital (pKa = 8.1) . Can your
finding be attributed to differences in their extent of ionization at pH
5.9 ?If not , how do you account for your experimental observations ?
7.
Discuss some of the advantages and disadvantages of utilizing the
goldfish membrane for studying the process of drug absorption .