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Simultaneous sampling of blood, bile, and urine in rats for pharmacokinetic studies
Simultaneous Sampling of Blood, Bile, and Urine in Rats for Pharmacokinetic Studies
SRIKUMARAN
ZHI-XIN Xv AND
MELETHIL
A method for simultaneous serial sampling of blood, bile, and urine from rats is described. Techniques for cannulation of jugular and femoral veins, bile duct, and bladder are described that make serial sampling of these three fluids possible. A saline infusion regimen was developed that prevents dehydration and maintains constant hematocrit values throughout the experiment. Accuracy of timed-samples can be easily controlled along with complete collection of voided urine. This method also allows for economy in terms of cost (number of animals used) and time in pharmacokinetic investigations involving the rat where extensive sampling of blood, bile, and urine are needed. In addition, interanimal variability, which can be quite high in such studies, is avoided by sampling all these three fluids in a single rat. While the kinetic parameters of blood aluminum obtained from this anesthetized animal model compare well with those in unanesthetized rats reported from this laboratory, this method is most useful for comparative studies.
Key Words:
Blood; Bile; Urine; Pharmacokinetics;
Hematocrit and Rat
INTRODUCTION Reasons of ready availability, popular experimental animal. along with blood are needed from
important
excretory
cost, and ease of maintenance
make the rat a very
Very often simultaneous sampling of bile and urine in pharmacokinetic studies, since these two routes
pathways
for drugs
and their
metabolites.
method for serial sampling of these three fluids is not available. deal with separate collection of blood (Popovic and Popovic, Weeks
and Davis,
19641, bile (Conway
and Melethil,
Oliver, 1964). In this report, an integrated method these three fluids is presented. The pharmacokinetic aluminum
in this model
obtained
in studies where
where
was carried
a
19741, and urine
(Cohen
and
for simultaneous sampling of parameters obtained for blood
the rat was anesthetized
sampling
However,
Published methods 1960; Upton, 1975;
compare
out in awake
well with values
animals.
EXPERIMENTAL Animals Male
Sprague-Dawley
light:l2-hr
From the Schools Address University Received
rats, weighing
dark cycle and housed reprint
of Pharmacy and Medicine, requests
to: Dr. Sri Melethil,
of Missouri-Kansas November
City, Medical
23,1989;
revised
350-400
g, were
maintained
two per cage. Rats were fed commercial University Division
School
of Missouri-Kansas of Pharmaceutical
Building,
2411 Holmes,
on a IZ-hr rat chow
City, Kansas City, Missouri. Science,
School
of Pharmacy,
Kansas City, MO 64108-2792.
and accepted March 6,199O.
203 Journal of Pharmacological 0 1990 Elsevwr
Science
Methods Publishing
24, 203-208 Co.,
Inc.,
0160.5402/90/$3.50
l1990)
655 Avenue
of ihe Americas,
New
York,
NY 10010
204
Z.-X. Xu and S. Melethil and tap water ad libitum. Animals or more prior to surgery.
were allowed
an acclimatization
period
of 4 days
Surgical Procedures Rats were
anesthetized
with
urethane
(Aldrich
a dose of 1.2 g/kg, i.p. The right jugular vein, femoral vein were cannulated in the mentioned tained Yellow
at 37°C using an automatic Springs,
abdomen.
OH).
feedback
control
The fur was shaved
The animal
Chemical
WI) at
inc., Milwaukee,
bladder, bile duct, trachea, and left order. Body temperature was main-
from
system (Yellow
Spring Co.,
the skin in front
was held on its back on the operating
Inc.,
of the neck and
table,
and a I-1.5-cm
incision was made with sharp scissors at the region above the midpoint of the right collar bone. The covering muscles and tissue were isolated by way of blunt dissection,
and a I-1.2-cm
silk thread
length
(size 5-O) were
of external
passed under
end of the vein, which was proximal
jugular
vein was exposed.
the blood
vessel and loosely
Two pieces of knotted.
to the head, was ligated first to prevent
during the cannulation procedure. An oblique cut was made on the anterior of the jugular vein, and a silicon polymer tubing (0.025 in. I.D.: 0.047 in. O.D., Corning
Corporation,
Midland,
of the heart and ligated jugular vein cannulation, atocrit
(Autocrit
MI) was placed
into the jugular
The
bleeding side Dow
vein in the direction
with the second knot. Immediately after completing the 50 ~J,Lof blood were withdrawn for measuring the hem-
Centrifuge,
Clay Adams,
Parsippany,
NJ). The cannulated
tubing
was then connected to an infusion pump (Harvard Apparatus Co., Inc., Millis, MA), and freshly made 0.9% sodium chloride solution was infused at 1.0 mlJhr. To cannulate
1 cm above
the su-
prapubic. The abdominal wall muscles were cut with scissors along with dominal white line in order to expose the bladder. Then, a small incision
the bladder,
the ab(2 mm)
was made
a 1.5-cm
length
at the tip of the bottom
incision
was made
of the bladder
while
about
it was held vertically
with
a pair of forceps. The bladder was then cannulated by a special bladder catheter made as follows prior to surgery. One inch of PE-200 tubing was slightly bent at the middle
using a hot plate to soften
total length
the tubing.
of 1 cm (0.5 cm on each side).
The bent tubing
Silicon
tubing
was cut to give a
(0.030 in I.D.:
0.065
in.
O.D.) was threaded through the bent PE-200 tubing so that about 1 cm of this silicon tubing was exposed at the other end. This shorter end, which had four holes on the tubing wall, was placed into the bladder via the incision, which was sealed with epoxy glue (Krazy Glue, Krazy Glue Inc., Itasca, IL) at the PE-200 sleeve. It was not possible to get a good seal between bladder tissue and silicon tubing. Suturing or ligation was avoided to prevent bleeding. The longer end of the tubing was then kept in a sample tube for urine collection. To expose the bile duct, an incision about 2.5 cm was made in the anterior abdominal wall under the xiphoid process. The facia and underlying muscles were cut with scissors along the abdominal white line. After exposure of the duodenum, the bile duct can be seen to emerge from the liver leading to the duodenum. After careful isolation of the duct from surrounding adipose tissue with forceps and cotton-tipped applicators, the bile duct was cut obliquely with fine sharp scissors (Fine Science Tools Inc., Belmont, CA), and a polyethylene (PE-20) tubing was inserted toward the liver and ligated with silk thread
Blood, Bile, and Urine Sampling in Rats
(size 5-O). The open end of the tubing was kept in a sample tube for bile collection. The abdominal wall was closed by suturing; the skin was closed with g-mm autoclips (Clay Adams, Parsippany, NJ) to prevent hypothermia and dehydration. A 1.5-cm length incision was then made along the midline of the anterior neck. By blunt separation of the muscles, the trachea was exposed, and a polyethylene tubing (PE160) was placed into the trachea. The tubing was anchored to the surrounding tissue with silk thread (size 5-O). The incision was finally closed with g-mm autoclips. Tracheotomy was performed to facilitate normal breathing and prevent animal death because of respiratory failure caused by secretory obstruction of the trachea. The left femoral vein was last cannulated. A 1.5-cm long incision was made in the groin area, and the left femoral vein was isolated by blunt dissection of the surrounding muscular tissue. A technique similar to that used for jugular vein cannulation was employed. Immediately after finishing the cannulation, 50 PL of blood was withdrawn to measure the hematocrit. The silicon polymer tubing was then filled with heparin solution (15 units/ml) to prevent blood from coagulating in the tubing. The whole procedure can be completed in about 1 hr. The abdomen, although not open, was covered with a gauze sponge that was kept moistened with a 0.9% sodium chloride solution. The hematocrit was measured at I-hr intervals for 6 hr, and then at 2-hr intervals for the remainder of the experiment. Dosing and Sampling Procedures After completion of the above surgical procedures, the animal was ready for dosing and serial collection of blood, bile, and urine. The aluminum dose was injected via the jugular vein, temporarily stopping the saline infusion, followed by flushing with 0.9% normal saline solution. Infusion of saline was restarted at 3 mL/hr for the next 2 hr (postdosing) and then decreased to 2 mL/hr for the remainder of the study (next 10 hr). The two groups of rats (3 in 0.1 mg/kg dose group and 4 in 1.0 mg/kg
TABLE 1
Hematocrit
(Femoral Vein) Values During the Experiment HEMATOCRIT(%)a-~~
TIME
No.
(HR)
1
2
3
4
5
6
Ob
46 46 47 48 47 47 46 46 46 45 46
45 46 45 47 44 43 46
47 48 47 48 47 48 48 49 47 48 47
44 43 45 43 43 44 42 43 44 42 44
44 44 43 45 43 44 42 41 43 42 41
43 43 45 43 43 43 42 42 42 42 40
0 1
2 3 4 5 6
a 10 12
43 46 45
a Saline infusion (3 mUhr for the first 2 h followed by 2 mUhr for the next 10 h). b Jugular vein blood (predosing).
MEAN f 44 45 45 46 44 45 44 44 44 44 44
k k k rt + 2 ? k k t *
SD 1.4 2.0 1.5 2.3 2.0 2.1 2.6 3.2 1.7 2.6 2.8
205
206
Z.-X.
Xu and S. Melethil
dose group) received either 0.1 or 1.0 mg/kg of aluminum as the sulfate salt. Blood samples (50 +L) were withdrawn at 0 (predosing), 1, 5, IO, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 300, 360, 480, 600, and 720 min, postadministration. Urine samples were collected at hourly intervals for the first 6 hr and at 2-hr intervals between 6 and 12 hr. Urine flow remained steady (0.5-0.7 mL/hr) during this collection period. Bile samples (0.8-1.0 mL/hr) were collected in polyethylene tubes prior to dosing (0.5 hr) and postdosing at 30-min intervals for the first 2 hr, hourly intervals between 2 and 6 hr, and then at 2-hr intervals between 6 and 12 hr. After collection of blood, bile, and urine, samples were analyzed for aluminum as described (Pai and Melethil, 1989). RESULTS
AND DISCUSSION
The silicon polymer tubing rather than polyethylene tubing was used in blood vessel cannulation and bladderostomy. This is due to its greater softness and flexibility, which prevent vessel damage and reduce irritation as described (Upton, 1975). One common reason of animal death during the experiment was respiratory system failure caused by insufficient ventilation. The easiest way to maintain normal ventilation was by tracheal cannulation, without which animals died in about 4 hr. In this study, the dose of urethane used kept the animals anesthetized during the experimental period. Following administration of urethane, the animals were ready for surgery in about 2-5 min, provided that the dose was injected i.p. The infusion regimen for saline (3 mL/hr for the first 2 hr followed by 2 mL/hr for the rest of the experiment) maintained normal hematocrit values (Table I). In preliminary studies, using 1 or 2 mL/hr of saline (through out the experiment), hematocrit increased (52%-56%) during the first 6 hr. Such increases in hematocrit are commonly observed in humans undergoing surgery because of the associated stress. In addition, this infusion regimen also enabled maintenance of constant body weight during the experimental period; body weights before and after the experiment were 385 t 15 g (n = 6) and 390 & 14 g (n = 6), respectively. In one test animal that did not receive any infusion of saline, the body weight decreased from 395 to 375 g in 12 hr. This loss is in agreement with volumes of bile (11.2 t 1.34 mL), urine (6.1 & 1.2 mL) and blood (about 1.7 mL) that were collected, along with invisible evaporation due to respiration (about 15 mL/kg/l2 hr). This value for loss caused by respiration was calculated based on human data (about 3 mL/kg/l2 hr) multiplied by 5 to correct for the difference in respiratory rate. The blood kinetic parameters (details will be published separately) obtained from this anesthetized animal model are similar (Table 2) to those reported in awake animals from our laboratories (Pai and Melethil, 19891, except for the volume of distribution at steady state (Vss) in the 0.1 mg/kg group.The recoveries of injected aluminum in bile in the previous study (Pai and Melethil, 1989, where only the bile duct was cannulated) and the present study (Table 3) were also essentially identical. The bile flow between these two studies were essentially identical (8.7 + 1.5 mL/ 12 hr versus 8.8 4 1.6 mL/12 hr, p > 0.05). These results showed that the extensive surgery associated with the present study did not affect bile flow or biliary aluminum
Blood, Bile, and Urine Sampling in Rats TABLE 2 Comparison of Blood Pharmacokinetic Anesthetized and Unanesthetized’ Rats
Parameters for Aluminum
in
DOSE (MC/
0.1 1.0
ML)
KG)
0.65
? 0.12
1.06
69.2
? 9.51’
40.7
2 12.2
2093
Present
0.92
i
0.02
0.75
47.0
+ 9.00’
44.4
*
7.88
2245
Previous”
0.29
& 0.03
2.37
55.9
*
8.47
17.4
+ 3.80
57121
2 14043
Present
0.36
I? 0.12
2.05
54.3
-r- 11.2
19.0
2 5.92
56391
2 19073
clearance;
ke, disappearance
constant;
Vss, volume
AUCI,
the blood
concentration
a Pai and Melethil, ’ Geometric
excretion.
area under
of distribution
at steady state;
2 442 2 356
Cl, apparent
curve from zero to infinity.
1989.
mean.
’ Significantly
ferent
VSS(MUKG)
(Hjb
Previousa
Abbreviations: blood
KE (H-l)
AUCI(NC*MIN/
CL(MML/HR/
Tl/Z STUDY
KC)
different
However,
between
urinary
recoveries
these two studies
has been reported by reducing
(p < 0.05).
to decrease
aluminum
was significantly
renal blood flow (Gumbleton
mean artery pressure
ruba et al., 1987).
of injected
(Table 3). This is not unexpected,
Consequently,
resulting
dif-
since urethane
et al., 1990), most likely
from o12-adrenoreceptor
urine flow was significantly
blockade
(Car-
(p < 0.05) decreased
with the flow being 6.1 t 1.2 mU12 hr in the present study (n = 6), as compared to 8.2 + 2.1 mL/12 hr in the previous study (Pai and Melethil, 1989), where the rats (n = 11) were
not anesthetized.
This resulted
in decreased
recovery
of aluminum
in the urine. Pentobarbital was found by us to affect urine output to a much less extent than urethane. However, the disadvantage of this choice is that the usual dose of pentobarbital redosing
2-3
(50
times during
mg/kg i.p.) maintained the experiment
its effect
is thus needed
for only about and increases
4-5
hr;
the risk of
animal death caused by overdosage. Amytal, which has been reported by others (Thomsen and Olesen, 1981) not to affect urine flow and inulin clearance, is an other
alternative.
studies would in blood
However,
require
kinetic
its use as an anesthetic
intermittent
parameters
dosing,
between
agent
in prolonged
as with pentobarbital.
anesthetized
and awake
RECOVERYOF Dos~(%/12
HR)
DOSE
0.1 1.0
URINE
BILE
STUDY Previousa
0.56
? 0.16
27.3
? 4.95’
Present
0.50
*
0.15
16.7
f
Previousa
0.19
f
0.072
18.3
? 5.06’
Present
0.21
? 0.061
a Pai and Melethil, b Significantly
1989.
different
(p < 0.05).
hr)
rats is due to the fact
TABLE 3 Biliary and Urinary Recoveries of Aluminum in the Rat”
(MC/KG)
(12-15
Lack of differences
4.61b
8.0 ? 2.83b
207
208
Z.-X. Xu and S. Melethil
that tissue-uptake plays a comparatively greater role in blood disappearance of aluminum than urinary excretion. Special care should be taken to perform bladderostomy without bleeding. The bottom tip of the bladder (free of blood vessels) was cut while it was held vertically with a forceps to prevent urine loss and bladder contraction upon cutting. Such contraction makes it difficult to insert the catheter and causes hemorrhage of submucosa capillaries, which can result in slight but sustained bleeding and subsequent contamination of urine samples. Four holes were made on the side of the silicon tubing inside the bladder for complete drainage of urine. In conclusion, determination of several pharmacokinetic parameters (e.g., blood, bile, and urine clearances) is made possible in a single rat by this method. Interanimal variation, a major concern in pharmacokinetic studies, is thus avoided. Simultaneous sampling also allows for economy in terms of cost (number of animals used) and time. Accuracy of timed-samples is easily controlled, particularly with respect to urine collection, which is commonly done by placing the rat in a metabolism cage, Such a procedure also does not allow urine collection over short intervals of time, which are needed when dealing with compounds that have short half-lives like aluminum. interestingly, aluminum disposition kinetics in animals was found to be essentially not influenced by anesthesia or extent of surgery in this study. While this may not be true with other compounds or drugs, this method is highly suitable for comparative pharmacokinetic studies in anesthetized rats.
REFERENCES Carruba MO, Bondiolotti G, Picotti GB, Catteruccia N, Prada MD (1987) Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat. Eur / Pharmaco/134:15-24.
Pai 5, Melethil S (1989) Kinetics of aluminum in rats I: Dose-dependent elimination from blood after intravenous administration. ) Pharmaceut SC) 78:200-203.
Cohen AE, Oliver HM (1964) Urethral catheterization of the rat. Lab Anim Care 14:471-473.
Popovic V, Popovic P (1960) Permanent cannulation of aorta and vena cava in rats and ground squirrels. ) Appi Physio) 15:727-728.
Conway WD, Melethil S (1974) Excretion of probenecid and its metabolites in bile and urine of rats. ) Pharmaceut Sci 63:1551-1554. Gumbleton M, Nicholls Pj, Taylor G (1990) Differential effects of anesthetic regimens on gentamicin pharmacokinetics in the rat: A comparison with chronically catheterized conscious animals. Pharm Res 7141-45.
Thomsen K, Olesen OV (1981) Renal Physio/4:165172. Upton RA (1975) Simple and reliable method for serial sampling of blood from rats. ) Pharmaceut SC; 64:112-114. Weeks JR, Davis JD (1964) Chronic intravenous cannulas for rat. ) Appl Physiol10:540-542.
SRIKUMARAN
ZHI-XIN Xv AND
MELETHIL
A method for simultaneous serial sampling of blood, bile, and urine from rats is described. Techniques for cannulation of jugular and femoral veins, bile duct, and bladder are described that make serial sampling of these three fluids possible. A saline infusion regimen was developed that prevents dehydration and maintains constant hematocrit values throughout the experiment. Accuracy of timed-samples can be easily controlled along with complete collection of voided urine. This method also allows for economy in terms of cost (number of animals used) and time in pharmacokinetic investigations involving the rat where extensive sampling of blood, bile, and urine are needed. In addition, interanimal variability, which can be quite high in such studies, is avoided by sampling all these three fluids in a single rat. While the kinetic parameters of blood aluminum obtained from this anesthetized animal model compare well with those in unanesthetized rats reported from this laboratory, this method is most useful for comparative studies.
Key Words:
Blood; Bile; Urine; Pharmacokinetics;
Hematocrit and Rat
INTRODUCTION Reasons of ready availability, popular experimental animal. along with blood are needed from
important
excretory
cost, and ease of maintenance
make the rat a very
Very often simultaneous sampling of bile and urine in pharmacokinetic studies, since these two routes
pathways
for drugs
and their
metabolites.
method for serial sampling of these three fluids is not available. deal with separate collection of blood (Popovic and Popovic, Weeks
and Davis,
19641, bile (Conway
and Melethil,
Oliver, 1964). In this report, an integrated method these three fluids is presented. The pharmacokinetic aluminum
in this model
obtained
in studies where
where
was carried
a
19741, and urine
(Cohen
and
for simultaneous sampling of parameters obtained for blood
the rat was anesthetized
sampling
However,
Published methods 1960; Upton, 1975;
compare
out in awake
well with values
animals.
EXPERIMENTAL Animals Male
Sprague-Dawley
light:l2-hr
From the Schools Address University Received
rats, weighing
dark cycle and housed reprint
of Pharmacy and Medicine, requests
to: Dr. Sri Melethil,
of Missouri-Kansas November
City, Medical
23,1989;
revised
350-400
g, were
maintained
two per cage. Rats were fed commercial University Division
School
of Missouri-Kansas of Pharmaceutical
Building,
2411 Holmes,
on a IZ-hr rat chow
City, Kansas City, Missouri. Science,
School
of Pharmacy,
Kansas City, MO 64108-2792.
and accepted March 6,199O.
203 Journal of Pharmacological 0 1990 Elsevwr
Science
Methods Publishing
24, 203-208 Co.,
Inc.,
0160.5402/90/$3.50
l1990)
655 Avenue
of ihe Americas,
New
York,
NY 10010
204
Z.-X. Xu and S. Melethil and tap water ad libitum. Animals or more prior to surgery.
were allowed
an acclimatization
period
of 4 days
Surgical Procedures Rats were
anesthetized
with
urethane
(Aldrich
a dose of 1.2 g/kg, i.p. The right jugular vein, femoral vein were cannulated in the mentioned tained Yellow
at 37°C using an automatic Springs,
abdomen.
OH).
feedback
control
The fur was shaved
The animal
Chemical
WI) at
inc., Milwaukee,
bladder, bile duct, trachea, and left order. Body temperature was main-
from
system (Yellow
Spring Co.,
the skin in front
was held on its back on the operating
Inc.,
of the neck and
table,
and a I-1.5-cm
incision was made with sharp scissors at the region above the midpoint of the right collar bone. The covering muscles and tissue were isolated by way of blunt dissection,
and a I-1.2-cm
silk thread
length
(size 5-O) were
of external
passed under
end of the vein, which was proximal
jugular
vein was exposed.
the blood
vessel and loosely
Two pieces of knotted.
to the head, was ligated first to prevent
during the cannulation procedure. An oblique cut was made on the anterior of the jugular vein, and a silicon polymer tubing (0.025 in. I.D.: 0.047 in. O.D., Corning
Corporation,
Midland,
of the heart and ligated jugular vein cannulation, atocrit
(Autocrit
MI) was placed
into the jugular
The
bleeding side Dow
vein in the direction
with the second knot. Immediately after completing the 50 ~J,Lof blood were withdrawn for measuring the hem-
Centrifuge,
Clay Adams,
Parsippany,
NJ). The cannulated
tubing
was then connected to an infusion pump (Harvard Apparatus Co., Inc., Millis, MA), and freshly made 0.9% sodium chloride solution was infused at 1.0 mlJhr. To cannulate
1 cm above
the su-
prapubic. The abdominal wall muscles were cut with scissors along with dominal white line in order to expose the bladder. Then, a small incision
the bladder,
the ab(2 mm)
was made
a 1.5-cm
length
at the tip of the bottom
incision
was made
of the bladder
while
about
it was held vertically
with
a pair of forceps. The bladder was then cannulated by a special bladder catheter made as follows prior to surgery. One inch of PE-200 tubing was slightly bent at the middle
using a hot plate to soften
total length
the tubing.
of 1 cm (0.5 cm on each side).
The bent tubing
Silicon
tubing
was cut to give a
(0.030 in I.D.:
0.065
in.
O.D.) was threaded through the bent PE-200 tubing so that about 1 cm of this silicon tubing was exposed at the other end. This shorter end, which had four holes on the tubing wall, was placed into the bladder via the incision, which was sealed with epoxy glue (Krazy Glue, Krazy Glue Inc., Itasca, IL) at the PE-200 sleeve. It was not possible to get a good seal between bladder tissue and silicon tubing. Suturing or ligation was avoided to prevent bleeding. The longer end of the tubing was then kept in a sample tube for urine collection. To expose the bile duct, an incision about 2.5 cm was made in the anterior abdominal wall under the xiphoid process. The facia and underlying muscles were cut with scissors along the abdominal white line. After exposure of the duodenum, the bile duct can be seen to emerge from the liver leading to the duodenum. After careful isolation of the duct from surrounding adipose tissue with forceps and cotton-tipped applicators, the bile duct was cut obliquely with fine sharp scissors (Fine Science Tools Inc., Belmont, CA), and a polyethylene (PE-20) tubing was inserted toward the liver and ligated with silk thread
Blood, Bile, and Urine Sampling in Rats
(size 5-O). The open end of the tubing was kept in a sample tube for bile collection. The abdominal wall was closed by suturing; the skin was closed with g-mm autoclips (Clay Adams, Parsippany, NJ) to prevent hypothermia and dehydration. A 1.5-cm length incision was then made along the midline of the anterior neck. By blunt separation of the muscles, the trachea was exposed, and a polyethylene tubing (PE160) was placed into the trachea. The tubing was anchored to the surrounding tissue with silk thread (size 5-O). The incision was finally closed with g-mm autoclips. Tracheotomy was performed to facilitate normal breathing and prevent animal death because of respiratory failure caused by secretory obstruction of the trachea. The left femoral vein was last cannulated. A 1.5-cm long incision was made in the groin area, and the left femoral vein was isolated by blunt dissection of the surrounding muscular tissue. A technique similar to that used for jugular vein cannulation was employed. Immediately after finishing the cannulation, 50 PL of blood was withdrawn to measure the hematocrit. The silicon polymer tubing was then filled with heparin solution (15 units/ml) to prevent blood from coagulating in the tubing. The whole procedure can be completed in about 1 hr. The abdomen, although not open, was covered with a gauze sponge that was kept moistened with a 0.9% sodium chloride solution. The hematocrit was measured at I-hr intervals for 6 hr, and then at 2-hr intervals for the remainder of the experiment. Dosing and Sampling Procedures After completion of the above surgical procedures, the animal was ready for dosing and serial collection of blood, bile, and urine. The aluminum dose was injected via the jugular vein, temporarily stopping the saline infusion, followed by flushing with 0.9% normal saline solution. Infusion of saline was restarted at 3 mL/hr for the next 2 hr (postdosing) and then decreased to 2 mL/hr for the remainder of the study (next 10 hr). The two groups of rats (3 in 0.1 mg/kg dose group and 4 in 1.0 mg/kg
TABLE 1
Hematocrit
(Femoral Vein) Values During the Experiment HEMATOCRIT(%)a-~~
TIME
No.
(HR)
1
2
3
4
5
6
Ob
46 46 47 48 47 47 46 46 46 45 46
45 46 45 47 44 43 46
47 48 47 48 47 48 48 49 47 48 47
44 43 45 43 43 44 42 43 44 42 44
44 44 43 45 43 44 42 41 43 42 41
43 43 45 43 43 43 42 42 42 42 40
0 1
2 3 4 5 6
a 10 12
43 46 45
a Saline infusion (3 mUhr for the first 2 h followed by 2 mUhr for the next 10 h). b Jugular vein blood (predosing).
MEAN f 44 45 45 46 44 45 44 44 44 44 44
k k k rt + 2 ? k k t *
SD 1.4 2.0 1.5 2.3 2.0 2.1 2.6 3.2 1.7 2.6 2.8
205
206
Z.-X.
Xu and S. Melethil
dose group) received either 0.1 or 1.0 mg/kg of aluminum as the sulfate salt. Blood samples (50 +L) were withdrawn at 0 (predosing), 1, 5, IO, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 300, 360, 480, 600, and 720 min, postadministration. Urine samples were collected at hourly intervals for the first 6 hr and at 2-hr intervals between 6 and 12 hr. Urine flow remained steady (0.5-0.7 mL/hr) during this collection period. Bile samples (0.8-1.0 mL/hr) were collected in polyethylene tubes prior to dosing (0.5 hr) and postdosing at 30-min intervals for the first 2 hr, hourly intervals between 2 and 6 hr, and then at 2-hr intervals between 6 and 12 hr. After collection of blood, bile, and urine, samples were analyzed for aluminum as described (Pai and Melethil, 1989). RESULTS
AND DISCUSSION
The silicon polymer tubing rather than polyethylene tubing was used in blood vessel cannulation and bladderostomy. This is due to its greater softness and flexibility, which prevent vessel damage and reduce irritation as described (Upton, 1975). One common reason of animal death during the experiment was respiratory system failure caused by insufficient ventilation. The easiest way to maintain normal ventilation was by tracheal cannulation, without which animals died in about 4 hr. In this study, the dose of urethane used kept the animals anesthetized during the experimental period. Following administration of urethane, the animals were ready for surgery in about 2-5 min, provided that the dose was injected i.p. The infusion regimen for saline (3 mL/hr for the first 2 hr followed by 2 mL/hr for the rest of the experiment) maintained normal hematocrit values (Table I). In preliminary studies, using 1 or 2 mL/hr of saline (through out the experiment), hematocrit increased (52%-56%) during the first 6 hr. Such increases in hematocrit are commonly observed in humans undergoing surgery because of the associated stress. In addition, this infusion regimen also enabled maintenance of constant body weight during the experimental period; body weights before and after the experiment were 385 t 15 g (n = 6) and 390 & 14 g (n = 6), respectively. In one test animal that did not receive any infusion of saline, the body weight decreased from 395 to 375 g in 12 hr. This loss is in agreement with volumes of bile (11.2 t 1.34 mL), urine (6.1 & 1.2 mL) and blood (about 1.7 mL) that were collected, along with invisible evaporation due to respiration (about 15 mL/kg/l2 hr). This value for loss caused by respiration was calculated based on human data (about 3 mL/kg/l2 hr) multiplied by 5 to correct for the difference in respiratory rate. The blood kinetic parameters (details will be published separately) obtained from this anesthetized animal model are similar (Table 2) to those reported in awake animals from our laboratories (Pai and Melethil, 19891, except for the volume of distribution at steady state (Vss) in the 0.1 mg/kg group.The recoveries of injected aluminum in bile in the previous study (Pai and Melethil, 1989, where only the bile duct was cannulated) and the present study (Table 3) were also essentially identical. The bile flow between these two studies were essentially identical (8.7 + 1.5 mL/ 12 hr versus 8.8 4 1.6 mL/12 hr, p > 0.05). These results showed that the extensive surgery associated with the present study did not affect bile flow or biliary aluminum
Blood, Bile, and Urine Sampling in Rats TABLE 2 Comparison of Blood Pharmacokinetic Anesthetized and Unanesthetized’ Rats
Parameters for Aluminum
in
DOSE (MC/
0.1 1.0
ML)
KG)
0.65
? 0.12
1.06
69.2
? 9.51’
40.7
2 12.2
2093
Present
0.92
i
0.02
0.75
47.0
+ 9.00’
44.4
*
7.88
2245
Previous”
0.29
& 0.03
2.37
55.9
*
8.47
17.4
+ 3.80
57121
2 14043
Present
0.36
I? 0.12
2.05
54.3
-r- 11.2
19.0
2 5.92
56391
2 19073
clearance;
ke, disappearance
constant;
Vss, volume
AUCI,
the blood
concentration
a Pai and Melethil, ’ Geometric
excretion.
area under
of distribution
at steady state;
2 442 2 356
Cl, apparent
curve from zero to infinity.
1989.
mean.
’ Significantly
ferent
VSS(MUKG)
(Hjb
Previousa
Abbreviations: blood
KE (H-l)
AUCI(NC*MIN/
CL(MML/HR/
Tl/Z STUDY
KC)
different
However,
between
urinary
recoveries
these two studies
has been reported by reducing
(p < 0.05).
to decrease
aluminum
was significantly
renal blood flow (Gumbleton
mean artery pressure
ruba et al., 1987).
of injected
(Table 3). This is not unexpected,
Consequently,
resulting
dif-
since urethane
et al., 1990), most likely
from o12-adrenoreceptor
urine flow was significantly
blockade
(Car-
(p < 0.05) decreased
with the flow being 6.1 t 1.2 mU12 hr in the present study (n = 6), as compared to 8.2 + 2.1 mL/12 hr in the previous study (Pai and Melethil, 1989), where the rats (n = 11) were
not anesthetized.
This resulted
in decreased
recovery
of aluminum
in the urine. Pentobarbital was found by us to affect urine output to a much less extent than urethane. However, the disadvantage of this choice is that the usual dose of pentobarbital redosing
2-3
(50
times during
mg/kg i.p.) maintained the experiment
its effect
is thus needed
for only about and increases
4-5
hr;
the risk of
animal death caused by overdosage. Amytal, which has been reported by others (Thomsen and Olesen, 1981) not to affect urine flow and inulin clearance, is an other
alternative.
studies would in blood
However,
require
kinetic
its use as an anesthetic
intermittent
parameters
dosing,
between
agent
in prolonged
as with pentobarbital.
anesthetized
and awake
RECOVERYOF Dos~(%/12
HR)
DOSE
0.1 1.0
URINE
BILE
STUDY Previousa
0.56
? 0.16
27.3
? 4.95’
Present
0.50
*
0.15
16.7
f
Previousa
0.19
f
0.072
18.3
? 5.06’
Present
0.21
? 0.061
a Pai and Melethil, b Significantly
1989.
different
(p < 0.05).
hr)
rats is due to the fact
TABLE 3 Biliary and Urinary Recoveries of Aluminum in the Rat”
(MC/KG)
(12-15
Lack of differences
4.61b
8.0 ? 2.83b
207
208
Z.-X. Xu and S. Melethil
that tissue-uptake plays a comparatively greater role in blood disappearance of aluminum than urinary excretion. Special care should be taken to perform bladderostomy without bleeding. The bottom tip of the bladder (free of blood vessels) was cut while it was held vertically with a forceps to prevent urine loss and bladder contraction upon cutting. Such contraction makes it difficult to insert the catheter and causes hemorrhage of submucosa capillaries, which can result in slight but sustained bleeding and subsequent contamination of urine samples. Four holes were made on the side of the silicon tubing inside the bladder for complete drainage of urine. In conclusion, determination of several pharmacokinetic parameters (e.g., blood, bile, and urine clearances) is made possible in a single rat by this method. Interanimal variation, a major concern in pharmacokinetic studies, is thus avoided. Simultaneous sampling also allows for economy in terms of cost (number of animals used) and time. Accuracy of timed-samples is easily controlled, particularly with respect to urine collection, which is commonly done by placing the rat in a metabolism cage, Such a procedure also does not allow urine collection over short intervals of time, which are needed when dealing with compounds that have short half-lives like aluminum. interestingly, aluminum disposition kinetics in animals was found to be essentially not influenced by anesthesia or extent of surgery in this study. While this may not be true with other compounds or drugs, this method is highly suitable for comparative pharmacokinetic studies in anesthetized rats.
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Pai 5, Melethil S (1989) Kinetics of aluminum in rats I: Dose-dependent elimination from blood after intravenous administration. ) Pharmaceut SC) 78:200-203.
Cohen AE, Oliver HM (1964) Urethral catheterization of the rat. Lab Anim Care 14:471-473.
Popovic V, Popovic P (1960) Permanent cannulation of aorta and vena cava in rats and ground squirrels. ) Appi Physio) 15:727-728.
Conway WD, Melethil S (1974) Excretion of probenecid and its metabolites in bile and urine of rats. ) Pharmaceut Sci 63:1551-1554. Gumbleton M, Nicholls Pj, Taylor G (1990) Differential effects of anesthetic regimens on gentamicin pharmacokinetics in the rat: A comparison with chronically catheterized conscious animals. Pharm Res 7141-45.
Thomsen K, Olesen OV (1981) Renal Physio/4:165172. Upton RA (1975) Simple and reliable method for serial sampling of blood from rats. ) Pharmaceut SC; 64:112-114. Weeks JR, Davis JD (1964) Chronic intravenous cannulas for rat. ) Appl Physiol10:540-542.