Shenyang Pharmaceutical University
LAB 20: INTESTINAL ABSORPTION
PHARMACEUTICS II
LABORATORY 20: In situ intestinal absorption experiment in
rats
1. LABORATORY OBJECTIVES
a) To learn how to conduct the in situ intestinal absorption experiment in rats.
b) To learn how to calculate the first-order absorption rate constant (Ka) and the
absorption percentage per hour.
2. INTRODUCTION
Most drugs pass through biomembranes from the high concentration side to the low
concentration side via passive diffusion. Passive diffusion can be described by the Fick’s
Law (Eq. 1), in which the diffusion rate (dC/dt) is proportional to the concentration
difference between the two sides of the cellular membrane.
-dc/dt=Ka△C=Ka(C-Cb)
(1)
where C is the concentration of drug in the gastrointestinal (GI) tract, Cb is the
concentration of drug in blood and Ka is the absorption rate constant. The diffusion rate
(dC/dt) can be influenced by the diffusion coefficient of the drug, the membrane thickness
and area, and the drug membrane permeability.
Gastrointestinal absorption is a process where a drug diffuses from the gastrointestinal
side (absorption site) to the blood side. Since the drug entering the blood will be quickly
distributed throughout the body, the drug concentration in blood will be very low compared
with that at the absorption site. Therefore, the blood compartment can be considered to be
under “sink condition” after oral administration of most drugs. This has resulted in a large
concentration gradient across the GI membrane during the entire absorption process. That
is, C>>Cb, so ∆C≈C, and Equation 1 can be rewritten as:
-dc/dt = KaC
(2)
Equation (2) is consistent with the standard first-order kinetics. The absorption of most
drugs from the gastrointestinal tract follows first-order kinetics. On the other hand, the
membrane diffusion rate can be described as the reduction in the amount of drug at the
absorption site (dXa/dt):
-dXa/dt = KaX
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(3)
Shenyang Pharmaceutical University
LAB 20: INTESTINAL ABSORPTION
PHARMACEUTICS II
Integrating equation 3 and then transforming it into the corresponding logarithmic form:
LnXa = lnXa(0)-Kat
(4)
where Xa is the amount of drug in the gastrointestinal tract, Xa(0) is the amount of drug in
the gastrointestinal tract at t=0, Ka is the drug absorption rate constant. Plotting lnXa
versus t gives a straight line with the slope equal to Ka.
3. METHODS
3.1 Apparatus
Peristaltic pump; UV spectrophotometer; infrared light source; surgical scissors
hemostat; latex tubing; beaker; fixing plate; electrically heated thermostatically-controlled
water bath.
3.2 Reagents
(1) 0.1% NaNO2 solution, (2) 0.5% NH2SO3NH4 solution, (3) 0.1% Naphthalene
ethylenediamine dihydrochloride solution, ( these three reagents should be stored in a
refrigerator after preparation), (4) 1 M HCl, (5) 0.2 M NaOH, (6) physiological sodium
chloride solution, (7) Krebs-Ringer solution (1000 ml solution contains 17.8 g NaCl, 0.35
g KCl, 0.37 g CaCl2, 1.37 g NaHCO3, 0.32 g NaH2PO4, 0.02 g MgCl2, and 1.4 g glucose),
(8) sodium pentobarbital (10 mg/ml, intra-peritoneal injection 0.4 ml per 100 g rat for
anesthesia), and (9) sulfadiazine (SD).
3.3 Procedures
3.3.1 Add 85 ml of test solution (containing SD 2 mg and phenol red 2 mg in 100 ml
Krebs-Ringer’s solution) to the beaker of the circulation system (Figure 20-1).
3.3.2 Male rats weighing about 200 g are fasted overnight (about 18 hours) with free
access to water before the experiment. Rats are then anesthetized with an
intraperitoneal injection of 10 mg/ml of sodium pentobarbital solution (0.4 ml per
100 g rat) and then placed on the fixing plate.
3.3.3 During the experiment, the rats are kept under an infrared light to maintain their
normal body temperature. The abdomen is opened by creating a midline longitudinal
incision of around 3 cm. Small openings are cut at the top of the duodenum and the
bottom of the ileum, and then glass tubes (diameter 0.5 cm) are inserted into the
intestinal tract and secured with string. The intestinal segment is flushed with 37ºC
isotonic saline solution and then connected to the perfusion apparatus. The surgical
area is covered with gauze which has been wetted with isotonic saline.
3.3.4 Start the peristaltic pump and perfuse the intestinal segment initially at a flow rate of
5 ml/min and then adjusted to 2.5 ml/min after 10 min perfusion.
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Shenyang Pharmaceutical University
LAB 20: INTESTINAL ABSORPTION
PHARMACEUTICS II
3.3.5 Take 1.5 ml of perfusate from the beaker (1 ml to measure the SD concentration and
0.5 ml to measure the phenol red concentration) at t=0. Then replace it with 2 ml of
phenol red solution (containing 20 μg phenol red per ml of Krebs-Ringer solution).
After this step, collect 1.5 ml samples every 15 min (1 ml and 0.5 ml each), and
replace with 2 ml phenol red solution. The purpose for the addition of phenol red is
to measure the amount of water absorbed by the intestine at different time points
since phenol red is considered not to be absorbed by the intestine.
Figure 20-1. The schematic diagram of the in situ intestinal absorption experiment in rats.
3.4 Quantitative method
3.4.1 Quantitation of SD
To 1 ml of SD in a tube, 5 ml of 1 M HCl and 1 ml of 0.1%NaNO2 are added in
sequence; the tube is shaken and allowed to stand for 3 min. Afterwards, add 1 ml of 0.5%
ammonium sulfamate to the tube, shake the tube, and allow to stand for 3 min. 2 ml of
0.1% naphthalene ethylenediamine is then added to the tube followed by shaking the tube
and setting the tube aside for 20 min. Finally, the absorbance of the sample is determined at
550 nm for the calculation of the SD concentration.
Preparation of blank control solution: Take 1 ml of the test solution (containing SD 2
mg, and phenol red 2 mg in 100 ml Krebs-Ringer solution) as the sample. The procedures
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Shenyang Pharmaceutical University
LAB 20: INTESTINAL ABSORPTION
PHARMACEUTICS II
are the same as those described above in section “3.4.1”, but without the addition of
naphthalene ethylenediamine reagent.
3.4.2 Quantitation of phenol red
Add 5 ml of 0.2 M NaOH to 0.5 ml of sample in a tube. After shaking, the
absorbance of the sample is determined at 550 nm.
Blank control solution: 0.2 M NaOH solution.
3.5 Establishment of a calibration curve
3.5.1 Calibration curve of phenol red
100 mg of phenol red standard is accurately weighed and placed into a 1000 ml
volumetric flask, then diluted with 1% sodium carbonate (Na2CO3) solution to 1000 ml to
give a 100 μg/ml stock solution. Different volumes of the standard stock solution (1, 2, 3, 4,
5, 6 ml) are placed in 10 ml volumetric flasks respectively and are diluted to 10 ml with
distilled water. The absorbance of these solutions is measured by UV spectrometry
according to section “3.4.2”, “Quantitation of Phenol Red” and the calibration curve is
constructed.
3.5.2 Calibration curve of SD
Preparation of stock solution: 10 mg of SD standard is accurately weighed and placed
into a 100 ml of volumetric flask, then diluted with distilled water to 100 ml to give a 100
μg/ml stock solution.
Establishment of calibration curve: Different volumes of the above SD standard stock
solution (2, 4, 6, 8, 10 ml) are placed into 10 ml volumetric flasks and diluted to 10 ml
with distilled water. 1 ml samples of the above solutions are treated as described in section
3.4.1 “Quantitation of SD” for measuring the absorbance and constructing the calibration
curve.
4. RESULTS AND DISCUSSION
Based on the equations shown in Table 1, plot the logarithm of the residual amount of
drug in the intestinal tract versus time, and then calculate the adsorption rate constant (Ka)
and absorption percentage per hour.
absorption percent per hour(%) 
(residue amount at t  0) - (residue amount at t  1 hr)
 100%
residue amount at t  0
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Shenyang Pharmaceutical University
LAB 20: INTESTINAL ABSORPTION
PHARMACEUTICS II
Table 1 Equations to calculate the in situ intestinal absorption in rats
Sampling
time (hr)
Before
SD
Absorbance
Ao
Concentration
C0
perfusion
0
A1
C1
0.25
A2
C2
0.5
A3
tn
An
C3
Cn
Phenol Red
Absorbance
Volume of test
Residue
solution (ml)
amount
Concentration
(μg)
A0'
C 0'
V0=85 ml
A1'
C1'
V1=
C 2'
V2 
A3'
C 3'
V3 
(V 2  1.5)C ' 2 40
C'3
P3=C3V3+1.5
An'
C n'
Vn 
(Vn1  1.5)C ' n 1 40
C'n
Pn  CnVn 
A2'
Po=85×C0
C ' 0 V0
C '1
P1=C1 V1
(V1  1.5)  C ' 1 40
P2=C2V2+1.5C1
C' 2
(C1+C2)
n 1
1.5 Ci
i 1
5. QUESTIONS
a) What is the key for success in conducting this experiment? What factors should be
considered when performing this experiment following the procedures?
b) How can the experimental apparatus be further improved?
(Shujun WANG)
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