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Nigerian Journal of Physiological Sciences
Physiological Society of Nigeria
ISSN: 0794-859X
Vol. 22, Num. 1-2, 2007, pp. 65-68
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Nigerian
Journal of Physiological Sciences, Vol. 22, No. 1-2, 2007, pp. 65-68
Serum Protein and Enzyme Levels
in Rats Following Administration of Antioxidant Vitamins During Caffeinated and
Non-Caffeinated Paracetamol Induced Hepatotoxicity
V. S. Ekam* and P. E. Ebong
Department of Biochemistry, College of Medical Sciences, University of Calabar, Calabar, Nigeria E-mail: ekvisa@yahoo.com Tel: +234 8053300565.
Received: 29/6/2007
Accepted: 24/7/2007
Code Number: np07011
Summary
The effects of caffeinated and
non-caffeinated paracetamol administration, with or without vitamins A and E
supplementation on the protein and enzyme levels in Wistar albino rats were
investigated using cafeinated paracetamol and paracetamol as caffeinated and
non-caffeinated paracetamol respectively, and water soluble acetic acid
derivatives of vitamins A and E. Serum AST, ALT and ALP levels (u/l)
significantly increased (P<0.05) following paracetamol administration.
Caffeination as well as administration of vitamins A and E caused significant
decreases (P<0.05) in AST and ALP levels in all test groups when
co-administered with paracetamol and in ALT level except in the cafeinated
paracetamol + Vitamin E group in which ALT and ALP level except in the
cafeinated paracetamol + vitamin E group in which ALT and ALP levels
significantly increased (P<0.05). Total serum protein level (g/100ml)
significantly increased following caffeination as well as during
co-administration of cafeinated paracetamol and Vitamin E; and significantly
decreased during co-administration of paracetamol and vitamin A. Paracetamol
administration without caffeination or supplementation with vitamin A and E can
therefore cause increases in serum liver enzymes that is suggestive of liver
necrosis which can be ameliorated to varying degrees by caffeine, vitamin A and
E.
Key
Words: Paracetamol,
liver enzymes, cafeinated paracetamol, vitamins A and E
Introduction
Paracetamol
or acetaminophen is a popular domestic analgesic and antipyretic for adults and
children. It is a major metabolite of the now obsolete phenacetin, with an
analgesic effect, which is similar to that of aspirin. In therapeutic doses,
acetaminophen is usually well tolerated and free from side effects and
interactions with other drugs. Its easy availability as well as ease of acquisition
to the public even without prescription has led to the increase in reported
cases of toxicity caused by paracetamol. The first reports of paracetamol
poisoning in humans describing hepatic necrosis provoked a series of animal
studies which demonstrated that acute centrilobular hepatic necrosis with
collapse of the reticulum framework could be produced in some species, although
there were differences in susceptibility in various species (Thomas, 1993).
In
adult humans, hepatotoxicity may occur after ingestion of a single dose of 10
to 15g (150 to 250 mg/kg) of acetaminophen; doses of 20 to 25g or more are
potentially fatal. Clinical indications of hepatic damage become manifest
within 2 to 4 days of ingestion of toxic doses. Plasma aminotransferases are
elevated and the concentration of bilirubin in plasma may be increased and
prothrombin time is prolonged. About 10% of poisoned patients who do not
receive specific treatment develop severe liver damage; of these, 10 to 20 %
eventually die of hepatic failure. Biopsy of the liver reveals centrilobular
necrosis with sparing of the periportal area (Hardman et at, 1996).
Co-administration
of paracetamol using fixed ratio drug combinations is increasingly gaining
popularity. 500mg paracetamol with 30mg caffeine preparations are increasingly
being introduced into the market in various trade names like cafeinated
paracetamol, Medik-55, Boska etc. Tablets containing paracetamol (325mg) plus
32.5mg of dextropropoxyphene (co-proxamol, Distalgesic) have been used to
provide an effective dose of both drugs as well as give a mild euphoriant
effect. Despite the benefit of rapid analgesic effect and the probable decrease
in toxicity, co-administration with these agents is still a cause of concern
(Laurence et al, 1997).
Experimental
evidence suggests that stress may contribute to degenerative disorders and
enhancement of tumor growth (Maier et al, 1994) which is partly
associated with reduced immune response as a result of micronutrient depletion.
Physiological stress has been reported to decrease plasma levels of vitamins A,
E, B6 and C (Louw et al, 1991). During the past several
years, major epidemiological studies have addressed the role of the antioxidant
vitamins A, C, and E in the protection against cardiovascular and malignant
diseases. A plausible conclusion from this emerging body of literature is that
nutrient deficiency increases the risk for disease, and replacement of such
deficiencies may be expected to confer benefits (Marcus and Coulston, 1996).
Materials and Methods
Forty-two
albino rats of wistar strain weighing between 200 to 300g were obtained from
the animal house of the Department of Biochemistry, university of Calabar. They were kept under constant environmental and nutritional conditions throughout the
experiment. The animals were allowed free feeding with a standard diet and
drinking water ad libitum. They were divided into seven experimental
groups, each consisting of six rats and treated as follows:
Group
1: Distilled water
Group
2: Paracetamol
Group
3: Cafeinated paracetamol
Group
4: Paracetamol + vitamin A
Group
5: Paracetamol + vitamin E
Group
6: Cafeinated paracetamol + Vitamin A
Group
7: Cafeinated paracetamol + Vitamin E
Cafeinated
paracetamoland paracetamol were administered as caffeinated and
non-caffeinated paracetamol, using oral doses of 171.43mg/kg. Vitamin E acetate
(Ephynal) and vitamin A acetate were administered as vitamins E and A using
oral doses of 4.286mg/kg and 1428.57 i.u/kg respectively. At the end of the treatment
period of 14 days, the animals were sacrificed after overnight fasting. Blood
was collected into plane screw cap bottles for serum collection: this was
allowed to stand for 2 hours for clotting to take place. The serum was removed
using a Pasteur pipette into another set of tubes, after spinning in a MSE
clinical centrifuge at 1000rpm for 5 minutes. Serum protein was estimated
using the Biuret method of Doninger et al., 1972.Alanine amino
transferase and Aspartate aminotransferase activities were determined by the
method of Mathieu et al., 1982. Alkaline phosphatase activity in serum
was estimated by kit method of Tietz, (1982); which is based on the measurement
of the rate of hydrolysis of phosphate esters.
Statistical Analysis
Students t test and
analysis of variance (ANOVA) were used to analyze the data. Values of
P<0.05 were regarded as significant.
Results
The total serum protein and the concentration of
some liver enzymes in serum were measured in rats undergoing treatments with
caffeinated and non-caffeinated paracetamol alone as well as those supplements
with vitamins A and E and in control rats. Enzymes assayed were Alanine
aminotransferase (ALT), Aspartate aminotransferase (AST) and Alkaline phosphate
(ALP). The effect of treatment on these parameters is shown in table 1. The
total serum protein and the concentrations of enzymes measured were observed to
vary significantly (p<0.05) among and within the test groups and the
control. From the results obtained, There was a significant increase (p<
0.05) in total serum protein of the groups on cafeinated paracetamol(7.53
± 1.07) and paracetamol + Vitamin E
(8.58 ± 0.81) as well as a significant
decrease in the group on cafeinated paracetamol + Vitamin A (4.66 ± 0.43) compared to the paracetamol group.
The protein content (g/100ml) was however significantly reduced (p < 0.05)
in the cafeinated paracetamol + Vitamin A group (4.66 ± 0.43) compared to the cafeinated paracetamol group (7.53 ± 1.07) and significantly increased (p <
0.05) in the paracetamol + Vitamin E group (8.58 ±
0.81) compared to the paracetamol + Vitamin A group (6.29 ± 1.05) compared to the cafeinated
paracetamol + vitamin A group.
The
concentration of ALP (U/L) in the paracetamol treated group (121.90 ± 10.62) was significantly higher (p <
0.05) than that of the control group (76.01 ±
2.81). The reductions in ALP (U/L) in the groups on cafeinated paracetamol,
paracetamol + Vitamin A, paracetamol + Vitamin E, cafeinated paracetamol +
Vitamin A and cafeinated paracetamol + Vitamin E were significant (p < 0.05)
compared to the paracetamol group. Co-administration of Vitamin A with
cafeinated paracetamol (59.93 ± 4.89)
produced a significant reduction (p < 0.05) in ALP (U/L) compared with the
cafeinated paracetamol group. There was however a significant increase (p <
0.05) in ALP (U/L) in the cafeinated paracetamol + Vitamin A group (59.93 ± 4.89).
There were significant differences (p < 0.05) in
ALT and AST (U/L) among and within the test groups. Paracetamol administration
produced significant increases (p < 0.05) in both AST and ALT (U/L) (28.28 ± 1.96 and 114.34 ± 0.55) compared to the control group (17.51 ± 1.42 and 73.81 ± 1.45). There were significant decreases (p<0.05) in AST and
ALT in the cafeinated paracetamol paracetamol + Vitamin A. cafeinated
paracetamol + Vitamin A groups compared to the paracetamol group. cafeinated
paracetamol + Vitamin E however produced a significant increase (p<0.05) in
ALT (U/L) (121.44 ± 1.82) compared to
the paracetamol group.
There was a significant
decrease (p<0.05) in ALT (U/L) in the cafeinated paracetamol + Vitamin A
group (73.61 ± 0.80) compared to the
cafeinated paracetamol group (89.00 ±
0.70) as well as a significant increase (p < 0.05) in the cafeinated
paracetamol + Vitamin E group compared to the cafeinated paracetamol group.
Co-administration of Vitamin E produced significant decreases (p < 0.05) in
AST (U/L) when administered with paracetamol and cafeinated paracetamol when
compared to the cafeinated paracetamol group. Administration of cafeinated
paracetamol + Vitamin E also produced a significant increase (p < 0.05) in
ALT (U/L) compared to the cafeinated paracetamol + Vitamin A group.
Table 1: Total serum protein and enzymes activities in rats given oral doses of
paracetamol or panadol extra with or without vitamin A and E.
Group |
Treatment |
Total Serum Protein
(g/100ml) |
AST (U/L) |
ALT (U/L) |
ALP (U/L) |
1 |
Control |
5.98 ± 0.65 |
17.51 ± 1.42 |
73.81 ± 1.45 |
76.01 ± 2.81 |
2 |
Paracetamol |
5.93 ± 0.68b |
28.28 ± 1.96a |
114.34 ± 0.55a |
121.90 ± 10.62a |
3 |
Panadol Extra |
7.53 ± 1.07* |
23.54 ± 0.84* |
89.00 ± 0.70*
|
87.97 ± 2.06* |
4 |
Paracetamol +
Vitamin A |
6.46 ± 0.86 |
20.57 ± 0.70* |
92.23 ± 0.53*
|
64.5 ± 3.40* |
5 |
Paracetamol +
Vitamin E |
8.58 ± 0.81* |
20.30 ± 0.72* |
83.35 ± 0.97*
|
68.87 ± 7.87* |
6 |
Panadol Extra +
Vitamin A |
4.66 ± 0.43* |
23.67 ± 0.96* |
73.61 ± 0.80* |
59.93 ± 4.89* |
7 |
Panadol Extra +
Vitamin E |
6.29 ± 1.05 |
18.83 ± 0.59* |
121.44 ± 1.82* |
84.75 ± 4.27* |
Values
presented as mean ± SD; n = 6; * p< 0.05 compared to the paracetamol
group, *p< 0.05 compared to the paracetamol group, a p< 0.05 compared to the control, b
p> 0.05 compared to control. Dose of paracetamol = 171.43 mg/kg, Dose of
paracetamol in cafeinated paracetamol =171.43mg/kg, Dose of caffeine in cafeinated
paracetamol = 10.286mg/kg, Dose of Vitamin A = 4.286 mg/kg, Dose of Vitamin
E = 1428.57 iu/ kg.
Discussion
Paracetamol administration produced a decrease in
total serum protein while caffeinated paracetamol as well as supplementation
with antioxidant vitamins A and E caused increases in total serum protein above
that of the control group. However, the co-administration of vitamin A and
paracetamol extra resulted in a decrease in total protein.
The commonest
enzymes employed as indicators of hepatocellular damage are the transaminase
enzymes (Aspartate aminotransferase; AST and Alanine aminotransferase; ALT) and
Alkaline phosphatase; ALP. Damage to the liver results in increase in their
activities in plasma. Increases in serum enzyme activities are roughly
proportional to the extent of tissue damage (Gaw et al, 1995). Serum
enzyme activities such as AST, ALT and
ALP were increased following paracetamol administration. Similarly, the
caffeination of paracetamol resulted in a reduction in the level of these
enzymes. Further reductions were observed following the administration of
vitamins A and E; however the administration of vitamin E alongside with
caffeinated paracetamol resulted in increased levels of ALP and ALT.
Increase in
liver enzymes in serum following paracetamol administration has earlier been
reported (Gaw et al, 1995 and Hardman et al, 1996). It has also
been reported that paracetamol could be bioactivated enzymatically by
cytochrome P450 2EI in both the liver and the kidney (Hu et al,
1993). Metabolic activation by cytochrome P450 and prostaglandin
synthase are known to catalyse the conversion of paracetamol to the reactive intermediate, N-acetyl
parabenzoquinoneimine (NAPQI) which is believed to play an important role in
paracetamol mediated toxicity (Raucy et al, 1989). Proinflammatory
cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin-1a,
that are released in response t paracetamol intoxication are thought to be
responsible for some pathlogical manifestations of paracetamol-induced
hepatotoxicity (Blazka et al, 1995).
The cumulative oxidative damage is likely one of the mechanisms
producing the hepatotoxic effects of paracetamol administration in this study.
The observed higher increase in the level of ALT over AST may be due to a
leakage of cytoplasmic enzymes into circulation as a result of inflammation of
the liver cells. The molecular mechanism by which the antioxidant vitamins, and
caffeine reduce liver damage maybe due to their ability to maintain liver cell
integrity even in the presence of a hepatotoxic agent such as paracetamol.
Conclusion
This study reveals that
vitamins A and E as will as caffeine have protective roles in the maintenance
of liver cell integrity even in the presence of a hepatotoxic agent such as
paracetamol.
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©Physiological Society of Nigeria, 2007
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