Download Effects of Phenobarbital and carbon tetrachloride on liver enzymes

Odhiambo et al. .…J. Appl. Biosci. 2012.
Effects of phenobarbital and carbon tetrachloride on liver enzymes
Journal of Applied Biosciences 56: 4097– 4107
ISSN 1997–5902
Effects of Phenobarbital and carbon tetrachloride on
liver enzymes
Odhiambo F., Chek J.B.L and Moro J.O.
Department of Biochemistry, University of Nairobi, P.O Box 30197-00100, Nairobi, Kenya.
Corresponding author: Odhiambo Felix Blair, email address: [email protected]
Original submitted in on 18th April 2012. Published online at www.m.elewa.org on August 31st 2012.
ABSTRACT
Introduction: Barbiturates and carbon tetrachloride (CCL4) are known to have effects on liver enzymes.
Moreover, barbiturates which are used as sedative hypnotics, anticonvulsants generally cause induction of
liver microsomal enzymes while CCL4 cause hepatoxity releasing various liver enzymes.
Objective: To determine the effects of Phenobarbital and carbon tetrachloride on some selected liver
enzymes.
Methodology and results: Rats treated with Phenobarbital had higher levels of activity of transaminases,
alkaline phosphatase and longer to gain writing reflex while CCl4 treated rats had reduced levels of the
mentioned enzyme and shorter time to gain writing reflex
Conclusion and application: Treatment with Phenobarbital needs continuous adjustment and regulation of
dosage by the attending clinician. A history of the patient or a therapeutic drug monitoring to confirm
toxicology. It was therefore concluded that barbiturates are inducers of liver enzymes while CCl4 is a liver
toxicant
INTRODUCTION
Barbituric acid is a malonylurea obtained by
condensation of urea and malonic acid. Barbituric
acid is not a sedative –hypnotic but its derivatives
(V N Sharma, 1999). Barbituric acid was first
synthesized on December 1864 by German
researcher Adolf Von Bayer (www.wikipedia).
Barbiturates have been classified mainly according
to the speed of onset of action and their duration of
action as ultra short acting, short acting,
intermediate acting, and long acting barbiturates.
Barbiturates and carbon tetrachloride (CCL4) are
known to have effects on liver enzymes (Sherlock
et al 1974). Further barbiturates which are used as
sedative hypnotics, anticonvulsants, generally
cause induction of liver microsomal enzymes
(Sherlock et al, 1974). The major enzymes induced
include the enzyme cytochrome p450, which is the
major enzyme involved in drug metabolism.
Another enzyme that increases is the gamma
glutamyl transferase (GGT) known to be induced
by the anti- epileptics or anticonvulsants for
example the Phenobarbitals (Srivastava et al,
2002). Hence with heavy intake of barbiturates,
levels of cytochrome p450 and GGT rise and this
will lead to an increase in the rate of drug
metabolism. As a result, drugs administered after
the body is exposed to barbiturates are expected
to metabolize faster due to increase in levels of
drugs metabolizing enzyme cytochrome p450.
When liver is exposed to CCl4 it gets damaged.
This may lead to increase in the levels of liver
enzymes in the serum as a result of the damage to
the liver cells. Hepatocytes, are therefore found
the serum
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Odhiambo et al. .…J. Appl. Biosci. 2012.
Effects of phenobarbital and carbon tetrachloride on liver enzymes
(Sherlock et al, 1974). By noting the serum level of
these enzymes; the level of liver damage can be
assessed through the estimation of Serum
oxaloacetate transaminase (SGOT) and serum
glutamate pyruvate transaminase (SGPT) levels.
Other enzymes such as lactate dehydrogenase
found in the liver are expected to rise. Hence using
good assay techniques the levels of these
enzymes can be determined by calculating specific
enzyme activity for each of them. Recent finding
have documented a continued use of barbiturates
as drugs of choice in anesthesia, treatment of
epilepsy, and as anticonvulsants. There was need
therefore to understand how barbiturates influence
MATERIALS AND METHODOLOGY
• Phenobarbital.
• Thiopental sodium.
• Carbon tetrachloride (CCl4).
• 2mM ketoglutaric acid.
• 1.0M hydrochloric acid.
• Citric acid.
• 200mM D-alanine.
• 200mM L-aspartate.
• 0.1 Phosphate buffer.
• Sodium hydroxide (0.4M and 0.02M).
• 2, 4-Dinitrophenyhydrazine.
• Glycine glycine.
Experimental animals: Albino rats of one year in age
with their weights as indicated in appendix VII.
Procedure
Administration of Phenobarbital to the rats: There
were two rats in each group.The administration of the
Phenobarbital
and
thiopental
was
given
intraperitonearly.
Group1: They were treated with Phenobarbital sodium
30mg/kg three weeks after every two days.
Group2:These were treated with Phenobarbital sodium
15mg/kg for 3 weeks after every two days.
Group 3: They treated with carbon tetrachloride (10%
in vegetable oil) 24 hours previously before treatment
with thiopental sodium.
Group 4: They treated with CCl4 (20%) in vegetable oil
24 hours previously before treatment with thiopental
sodium.
Group 5: This group was not treated with any drug for
three weeks and served as control groups for the
Phenobarbital treated rats.
Group 6: Served as controls for CCl4 treated rats.
the body system. This is only possible through
continuous studies ascertaining their long-term and
short-term effects in human and animal systems.
This study was therefore done to confirm the
previous findings and also to find out if they have
to be continuously used or other better alternatives
can be applied. Exposure of the liver to various
toxins in the current world has increased
tremendously, the intake of preserved foods are
contributing factors in to this debate of
hepatotoxicity. CCl4 used a model to understand
the basis of study of liver damage and
hepatotoxicity.
Preparation of serum: After successful injection of the
rats with Phenobarbital sodium and feeding with
CCl4solution, they were injected with thiopental sodium
to study the physical effects. They were then sacrificed
to secure blood from which serum was obtained. The
serum was obtained by living the blood to clot for about
20 minutes by putting it in ice. After clotting the blood
was then centrifuged for 20 minutes at 6000 rpm. The
serum was then decanted using Pasteur pipettes to get
pure non-haemolysed blood containing serum. The
serum was then put into labeled eppendorf tubes
according to their respective drug treatments. The
serum samples were kept frozen at –20º before caring
out enzyme assays.
Enzyme assay procedures
Enzyme assays principles: GGT catalyses the
transfer of gamma glutamyl group from gamma
glutamyl-p-nitroanilide to accept or glycylglycine
according to the following reaction
Gamma–l-Glutamyl-3-carboxy-4-nitroanilide+ Glycyl
glycine GGT Gamma –l-glutamyl-glycylglycine
+ 2-Nitro-5-aminobenzoic acid
The rate of 2-nitro –5amino benzoic acid formation,
when measured photometrically indicates and
proportional to the catalytic concentration of the
enzyme present in the sample. The assay procedure
for both the AST and ALT were based on the
colorimetric method whereby the reaction was allowed
to take place by adding their substrates, alpha
ketoglutarate and aspartic acid for AST and alpha
ketoglutarate and alanine for ALT. The reactions were
then stopped by dinitrophenyl hydrazine (DNPH)
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Odhiambo et al. .…J. Appl. Biosci. 2012.
Effects of phenobarbital and carbon tetrachloride on liver enzymes
reagent. DNPH reacts with pyruvate to form a brown
colored complex medium which is measured
calorimetrically (DNPH can react with all oxoic acids). It
gives a triose colour with pyruvate and oxaloacetate
than with alpha ketoglutarate as evidenced by relative
absorbance for pyruvate, OAA and 2-oxoglulurate
being 1, 0.55 and 0.33 respectively. Because of the
blink value, the reaction is carried out at the sub optimal
concentration of oxoglutarate).
Procedure of assay of gamma glutamyl-transferase:
One ml of the working reagent was put into a cuvette
then 0.1ml of the serum sample was added for both the
controls and test. The contents were then mixed,
waited for 1 minute and read the initial absorbance (A)
of the sample at 405 nm. The stop watch was started
and the absorbance was read at 1-minute interval for
three minutes.
Glutamate Pyruvate Transaminase (GPT) assays: A
calibration graph of units/L against the optical density
reading at 505 is required and this is used in
determination of enzyme activity in sera the following
set up was used to generate the standard curve was
arranged as below:
Table1: Glutamate pyruvate assay Layout
Tube no.
Distilled water
Pyruvate standard (mls)
(mls)
1
0.2
0
2
0.2
0.05
3
0.2
0.1
4
0.2
0.15
5
0.2
0.20
6
0.2
0.25
7
0.2
0.30
After arranging the tubes as above, the contents were
then mixed well and incubated at 370 for 30 minutes.
After which 1 ml of 2, 4- Dinitrophenylhyrazine reagent
in 1M hydrochloric acid was added to each tube, mixed
well and incubated for further 20 minutes. 10 ml of 0.4M
sodium hydroxide solutions was added to each tube
and allowed to stand for 10 minutes. The optical
densities were read at 505 nm.
Assay Alanine Transaminase (ALT): One ml of the
buffered substrate was first put in the test tubes for the
assay of both test serum and control serum. Of the test
serum 0.2ml was added and incubated for 30minutes
at 37ºC, 1ml of 2,4 DNPH was added to each of the
tubes and 0.2 ml of the control serum was the added
its respective tube. The contents were then mixed well
GPT Substrate buffer
(ml)
1.0
0.95
0.90
0.85
0.80
0.75
0.70
Corresponding IU /L
0
6.5
12.5
21.0
30.5
39.5
56
and incubated further for 20 minutes then removed
from the water bath and 10ml of 0.4M NaOH added to
each tube. The tubes were left to stand for 10 minutes
after which the optical densities were read at 505 nm
against the Water blank.
The AST activity in serums was assayed as follows:
After arranging the tubes as above, the contents were
then mixed well and incubated at 37º for 30 minutes in
water bath. After which 1 ml of 2, 4Dinitrophenylhyrazine reagent in 1.M hydrochloric acid
was added to each tube, mixed well and incubated for a
further 20 minutes. Ten (10) mls of 0.4M sodium
hydroxide solutions was added to each tube and
allowed to stand for 10 minutes. The optical densities
were read at 505 nm.
Table 2: Aspartate aminotransferase assay layout.
Tube no.
Distilled water
Pyruvate standard
(Mls)
(mls)
1
0.2
0
2
0.2
0.05
3
0.2
0.1
4
0.2
0.15
5
0.2
0.20
6
0.2
0.25
7
0.2
0.30
4099
AST substrate
buffer (ml) Buffer
1.0
0.95
0.90
0.85
0.80
0.75
0.70
Corresponding IU /L
0.0
6.0
13.0
22.0
30.5
39.5
51.0
Odhiambo et al. .…J. Appl. Biosci. 2012.
Effects of phenobarbital and carbon tetrachloride on liver enzymes
The Procedure for the assay of serum AST levels
was as follows: One ml of the buffered substrate was
first put in the test tubes for assay of both test serum
and control serums. Of the test serum 0.2 ml was
added into the test tube and all the tubes incubated at
37º C for 1 hour in water bath. A drop of aniline citrate
was added in each tube. The samples were left in water
bath for 5 minutes, still at 37º C. Of the control serums
0.2 ml was then added followed by addition of 1ml of 2,
4 DNPH additions to all tubes. The contents were
mixed and incubated further for 15 minutes and then
removed. 10ml of 0.4M sodium hydroxide was added to
the reaction. The contents were mixed and allowed to
stand for ten minutes. The optical density was at 505
nm against water blank.
Preparation of the calibration graph for the alkaline
phosphatase assay: The tubes were arranged as
follows then filled with the reagent therein.
Table 3: Assay of alkaline phosphatase
Tube No
1
2
3
4
5
6
Alkaline Phophatase
4 - Nitrophenol Solution
0.5
1
1.5
2.5
3.5
4.5
The contents were then mixed well and their
absorbance read at 400nm against water blank.
Assay activity of the alkaline phosphatase in
serum: Two test tubes were prepared for each serum
to be assayed i.e. both the test and control serum 1.0ml
of the buffered substrate was put into each of the tubes.
Test (here for both the control and the test serum) and
0.02M NaOH
10.6
10.1
9.6
8.6
7.6
6.6
the serum blank. This was then incubated for 37º C for
5 minutes and serum added only in the tube for test.
Mixed well and incubate at 37ºC for 30 minutes
including serum blank. After that 10ml 0.02M NaOH
was added. Serum was then added in the serum blank
tubes. The contents were then mixed well in each tube
and the optical densities read at 400nm.
RESULTS AND DATA ANALYSIS
Table 4: Calculated values of Gamma Glutamyl-transferase activity after treatment with Phenobarbital and carbon
tetrachloride.
Test Group
Rat
OD
Factor
GGT activity (U/L)
1
a
0.012
1190
14.28
b
0.0096
1190
11.424
2
a
0.007
1190
8.33
b
0.014
1190
16.66
3
a
0.019
1190
22.61
b
0.014
1190
16.66
4
a
0.015
1190
17.85
b
0.0172
1190
20.468
5
a
0.006
1190
7.14
b
0.0095
1190
11.305
6
a
0.0034
1190
4.046
b
0.0055
1190
6.545
4100
Figure 1: Calibration Graph for the determination of GOT activity.
Figure 2: Calibration curve for determination ALT activity
4101
Figure 3: Calibration curve for alkaline phosphatase
Table 5: Activity for Aspartate aminotransferase after treatment with Phenobarbital and carbon tetrachloride
Test Group
Rat
OD
AST activity (U/L)
1
a
0.035
45
b
0.033
32.5
2
a
0.0352
40
b
0.0278
18
3
a
0.038
55
b
0.032
29.5
4
a
0.026
14
b
0.036
44.5
5
a
0.022
8
b
0.024
10.5
6
a
0.025
12.5
b
0.021
4.5
Table 6: Activity for Alanine aminotransferase after treatment with Phenobarbital and carbon tetrachloride.
Test Group
Rat
OD
ALT activity (U/L)
1
a
0.303
17
b
0.321
17.5
2
a
0.238
10.5
b
0.332
17.6
3
a
0.452
27.5
b
0.341
18
4
a
0.401
22.5
b
0.449
25
5
a
0.195
7.5
b
0.151
4
0.16
b
0.251
11
4102
Table 7: Results of the estimated activity of alkaline phosphatase after treatment with Phenobarbital and carbon
tetrachloride
Group
Rat
Test (OD)
Serum blank (OD)
OD difference
Enzyme activity
1
2
3
4
5
6
a
b
a
b
a
b
a
b
a
b
a
b
0.596
0.613
0.693
0.672
0.916
0.769
0.705
0.843
0.622
0.674
0.608
0.643
0.561
0.574
0.678
0.601
0.605
0.644
0.569
0.598
0.573
0.619
0.563
0.604
4103
0.035
0.039
0.015
0.071
0.311
0.125
0.136
0.245
0.049
0.055
0.045
0.039
15
16.5
6
28.5
130
52.5
37
103
21
23
18
16.5
Thiopental sodium
Thiopental sodium
Thiopental sodium
Thiopental sodium
Thiopental sodium
Thiopental sodium
Thiopental sodium
Thiopental sodium
Thiopental sodium
2b
3a
3b
4a
4b
5a
5b
6b
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
0.0 mins
6secs
25 secs
9secs
10 secs
4 secs
2mins
5secs
10secs
4mins 12 secs
5mins 23 secs
3mins 54 secs
58 secs
45secs
1 min 5 secs
54secs
Never regained
2hrs 13 mins
Never regained
3hrs 48 mins
19mis 29 secs
15mins10 secs
17mis 20 secs
42 mins
56mins 49 secs
1 hr 10 mins
1 hr 25 secs
Never recovered
2 hours 30mins
Never recovered
4hours 20 mins
23mins
18mins 12secs
3 mins 54 secs-5 mins 23secs
4secs-2 mins
6-25 secs
Phenobarbital
CCl4
Control
4104
45 secs- 1 mins 5 secs
2.5hrs- never
10-20mins
42mins- 1 hr 10 mins
18-35 mins
Table 9: Summary of the result on the effects of thiopental sodium treatment on rats after treatment with Phenobarbital and carbon tetrachloride.
Group of rat according to treatment
Time of loss of righting reflex
Time of regaining righting reflex
Time of recovery (walking)
Summary of the above results
6a
Thiopental sodium
2a
30 mg
36mins
Thiopental sodium
20.00mins
1b
4.0mins
35mins
0.0 mins
Thiopental sodium
1a
30 mg
Time of recovery
(walking)
Table 8: The effects of thiopental sodium treatment on rats after treatment with Phenobarbital and carbon tetrachloride
Rat
Drug
Dose per kg weight
Time of administration
Time of loss of
Time of regaining
righting reflex
righting reflex
DISCUSSION
Barbiturates are known to be enzyme inducers while
CCL4 is a hepatotoxicant able to cause liver
damage(.Levels of GGT in the serum can therefore be
used as an indication of either enzyme induction or liver
damage, the later being more common(. The GGT
levels also helps to detect liver damage and bile injury
while in some cases it is used in people suspected of
having liver diseases. It is also used to help explain the
cause of other changes in the liver or if alcohol abuse is
suspected Lab test online.org (2001-2008). For
example both ALP and GGT are elevated in diseases
of bile ducts and in some liver diseases but only ALP
will be elevated in bone diseases .If the GGT levels are
normal in a person with high ALP, the cause most likely
is bone disease . GGT can be used to screen for
chronic alcohol abuse where it will be elevated in about
75% of chronic diseases (Lab test online.org (20012008). The normal levels range form 4-18U/Lin males
and in females 6-28U/L when the assay is done at
room temperature (25ºC), )(Chrono lab AG Switzerland,
2008) From the results obtained, the rats that were not
treated (controls) had lower levels of the GGT activity
as compared to the ones treated with barbiturates.
Although the GGT levels in rats treated with
Phenobarbital were within the normal reference range,
their activity was higher than the controls and this could
be as a result of the induction made by the barbiturate
(Phenobarbital). Rat 2 (b) treated with 15mg/kg of the
drug had increased levels than others treated with the
same drug including the one treated with higher dosage
(30mg/kg) of the same drug. This could suggest other
factors that could induce the levels of GGT that could
be bone disease, bile duct injury or billiary obstruction.
But this are just suggestive as this could be combined
with other liver function tests involving other enzyme for
example higher levels of GGT and lower levels of ALP
rules out bone disease.
In hepatocellular damage higher levels of GGT have
always been observed (Lallit et al). In this case , rats
treated with CCL4 had increased levels, some even
beyond the normal documented levels of 4-18 U/L in
males (Chorono lab AG Switzerland, 2008), meaning
that CCL4 was responsible for the increased levels of
GGT for both treated with 20% and 10 % CCL4 in
vegetable oil. From the results obtained it was evident
that Phenobarbital is a good inducer of liver enzymes,
in rat 1a with an estimated level of 17U/L, which is
slightly above the normal levels. Rat 1b also had
increased levels of ALT of 17.5 U/L. From this it,
suggests that there was no hepatobilliary injury since
the levels of GGT was falling in a normal range among
these rats. Bone disease could also not be suggestive
compared to the results of ALP levels among these
rats, which were at normal levels. Rats in group two
also had increased elevated levels too, though rat 2a
was within the normal levels but on the higher side. Rat
2b had elevated levels above the normal range. This
also confirmed the levels in rats of group one. Rats in
group 3 and 4 had increased levels of both the
transaminases above their normal ranges. This could
not be due to induction but injury to the hepatocytes but
when compared to the levels of GGT it could confirm
that this rise in levels of transaminase was not due to
induction but due to liver damage. ALT levels of rats of
groups 5 and 4 fell within the normal range with rat 5b
having the minimal value of the enzyme level. ALT is
primary liver enzyme and its induction correlates to the
very effect of these drugs.
Serum level of AST, which is one of the enzymes most
used in diagnosis of liver function test, is also
suggestive of the pathologies of the liver. The rats in
group 1 treated with 30mg/kg had induced levels of
AST with level of rat 1a going up to 45 U/L much above
the maximum normal level maximum of 20U/L. Rats in
group in two also had increased levels of the enzyme
with 2a being up to 40 U/L. Even though 2b had 18U/L,
which falls within the normal range but on the higher
side. Rats of group 3 had excessive levels with 3a
going up to 55U/L and 3b 29.5U/L which when
compared to the reference range was high. This was
due to liver damage caused by the CCL4. Group 4
animals especially had elevated levels and explanation
was just as the group 3 rats. Rat 4a had normal levels.
May be there was no damage to it or the experiment
didn’t give a cognitive figure but it is believed it also
underwent liver damage as Levels of GGT indicates.
On the other hand, the effect of Phenobarbital and CCl4
on rat liver enzymes can be analyzed by physical
examination of rats by treating them with a short acting
barbiturate which can give immediate anesthetic effect
(Brodie et al, 1958). In this case thiopental sodium was
used. Rats on treatment with enzyme inducing agent
such as barbiturates (Phenobarbital in this case) take
shorter time to recover while those intoxicated with very
active hepatotoxicants take too long to recover or might
end up in dying Brodie et al, 1958). In this particular
study group 1 and 2 rats treated with Phenobarbital
previously, took longer time to lose their righting reflex,
between 3 mins 54 secs to-5 mins 23 secs. While those
on treatment with CCL4 took a shorter time (4 sec-2
4105
mins) due to enzyme induction and liver toxicity
(damage) respectively. Comparing the time of loss of
righting reflex with the control rats (6 secs-25secs)
gives an indication of barbiturate and CCL4 effects.
Rats treated with Phenobarbital took a shorter time (10
to 20 mins) to regain their righting reflex as compared
with those treated CCL4 which took longer time to
regain their righting reflex ranging from 2 hours for rat
4b and 4 hrs 20 mins for rat a. Others treated with CCL4
CONCLUSION AND RECOMMENDATIONS
From the results obtained from this experimental
analysis, it was important to conclude that barbiturates
are inducers of liver serum enzymes, which move to the
blood in excess. This was shown through the serum
enzyme analysis and evident in the results. It was
established that Phenobarbital, a barbiturate induces
liver enzymes. It was also found that CCL4 leads to
increase in the levels of serum enzymes but this was
due to liver damage it causes. There was evidence that
treatment with Phenobarbital leads to a shorter action
of thiopental sodium. This could suggest tolerance
obtained by these animals after continuous
administration. Patients therefore who have been on
treatment with drugs (barbiturates) need to increase
their dosage in order to get treated with other drugs to
the due to the induction of microsomal enzymes
increases. It was also confirmed that CCl4 is a liver
toxicant with severe hepatic damage according to the
enzyme levels of obtained and also after treatment with
thiopental sodium which indicated long time of gaining
the righting reflex even lack of recovery leading to
death. Patients, who have been on treatment with
Phenobarbital especially the epileptics, need a
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