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Nigerian Journal of Physiological Sciences
Physiological Society of Nigeria
ISSN: 0794-859X
Vol. 22, Num. 1-2, 2007, pp. 75-81
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Nigerian
Journal of Physiological Sciences, Vol. 22, No. 1-2, 2007, pp. 75-81
Effect of Caffeine -Coconut Products Interactions
on Induction of Microsomal
Drug-Metabolizing Enzymes in Wistar Albino Rats
A. E. , Abara1, G. O. Obochi,1 S. P. Malu1, M. Obi-abang1, V. S. Ekam2, and F. U. Uboh2.
1Department
of Biochemistry, Cross River University of Technology, Calabar,
2Department
of Biochemistry, University of Calabar, Nigeria. E-mail:
gobochi@yahoo.com Tel. +234 805 270 7200
Received: 22/5/2007
Accepted: 9/8/2007
Code Number: np07013
Summary
Effect of
caffeine-coconut products interactions on induction of drug-metabolizing enzyme
in wistar albino rats was studied. Twenty rats were randomly divided into four
groups: The control group (1) received via oral route a placebo (4.0ml of distilled
water). Groups 2 to 4 were treated for a 14-day period with 50mg/kg body weight
of caffeine, 50mg/kg body weight of caffeine and 50mg/kg body weight of coconut
water, and 50mg/kg body weight of caffeine and 50mg/kg body weight of coconut
milk in 4.0ml of the vehicle via gastric intubation respectively. One day after
the final exposure, the animals were anaestheticized by inhalation of an
overdose of chloroform. The blood of each rat was collected by cardiac
puncture while the liver of each rat was harvested and processed to examine
several biochemical parameters, ie, total protein and RNA levels, protein/RNA
ratios, and activities of alanine and aspartate amino transferase (ALT and AST,
respectively). The results showed that while ingestion of coconut milk and
coconut water increased the values of protein and protein/RNA ratios, it
decreased alanine and aspartate amino transferase (ALT and AST) activities.
These effects, in turn, enhanced the induction of the metabolizing enzymes and
a resultant faster clearance and elimination of the caffeine from the body,
there by reducing the toxic effect on the liver.
Key Words:
Coconut water, coconut milk, caffeine, protein, RNA, aminotransferases, liver and
enzymes.
Introduction
Humans are
widely exposed to various foreign chemicals such as drugs, food additives and
pollutants. These substances interfere with the absorption, metabolism,
distribution and excretion of nutrients, and are mostly insoluble in the body
fluids, thus, potentiating their toxic effects in tissues such as the liver and
brain (Eteng et al, 1997; Ekam, 2001; Obochi, 2006). Genetics,
environmental and physiological factors are involved in the regulation of drug
biotransformation reactions and are thought to be responsible for prolonged
pharmacological effects in the tissues (Bahtnager and Misra, 1988; Coon, 1996;
Raisfield et al, 2000; Obochi, 2006). Drugs metabolizing activities
within the cells take place primarily in the endoplasmic reticulum and involve
microsomal enzymes or mixed function oxidases (Goldberg and Mitsugi, 1967;
Craft et al, 1979; Bahtnager and Misra, 1988; Coon, 1996; Singh et al,
1998). These enzymes act to increase microsomal membrane phospholipids and
proteins, resulting in the proliferation of the smooth endoplasmic reticulum,
an adaptive measure, to increase the volume (hypertrophy) of the smooth
endosplasmic reticulum, and modification of these enzymes due to demethylation
process (Suresh and hedge, 1971; Bahtnager and Misra, 1988; Singh et al, 1998).
The significance of these enzymes modification is to increase the solubility or
hydrophilic properties of these foreign chemicals thereby facilitating their
excretion (Suresh and Hedge, 1971; Effiong, 2003; Obochi, 2006).
Caffeine,
1,3,7-trimethylxanthine, is consumed through processed cocoa, coffee, tea or
kola nut based foods and beverages, stimulants, drugs and cosmetics (Eteng et
al, 1997; Obochi, 2006). The metabolism and toxicity of caffeine are viewed
to be dose dependent, resulting in non-linear accumulation of the
methylxanthines, hence greater risk of cardiovascular diseases such as heart
failure, high blood pressure and neuronal disorder such as schizophrenia
(Cucinnel et al, 1965; Shaffner and Popper, 1969; Leon and Hedge, 1970).
Metabolic derangement and subsequent toxicity of caffeine leads to weight loss,
poor growth, low protein efficiency ratio and poor nitrogen retention, leading
to death (Leon and Hedge, 1970; Eten et al, 1997, Ekam, 2001). Thus, the
toxicity of caffeine is viewed as a disposition demonstrating that caffeine is
rapidly absorbed but slowly excreted (Leon and Hedge, 1970; Eteng et al, 1997;
Ekam, 2001; Obochi, 2006).
Caffeine
also acts to increase alertness, anxiety and hallucination due to its blocking
of adenosine receptors which normally inhibits glutamate release (Ekam, 2001;
Obochi, 2006). Glutamate supplies the amino group for the biosynthesis of amino
acids, and is a substrate for glutamine and glutathione synthesis (Danbolt,
2001). Inhibition of glutamate release results in low protein synthesis and
efficiency (Bertolini et al, 1980; Bahtnager and Misra 1988). Adenosine
receptors are linked with an interplay of release, reuptake, metabolism and
excretion of neurotransmitters (Obochi, 2006). Thus, blockage of adenosine
receptors by caffeine results in alteration in behavioural pattern by delaying
neuronal tube closure (Eteng et al, 1997; Obochi, 2006). Caffeine also
activates phosphorylase and lipase, thus, enhancing glucogenolysis and
lipolysis, resulting in loss of weight. Caffeine also inhibits androgen
binding protein, resulting in decreased cauda epididymis sperm reserve,
seminiferous tubular fluid volume, resulting in low sperm production and
infertility (Eteng et al, 1997; Obochi, 2006).
Mechanisms of action of caffeine involve
interaction with hormones, peptides and receptors on the surface of the plasma
membrane to generate signals since these molecules cannot cross the plasma
membrane (blood-brain-barrier). The amplification and subsequent transmission
of such signals to the cell interiors require the participation of second
messenger, usually a cyclic nucleotide, cAMP, while the hormones, peptides or
receptors serve as the first messenger (Eteng et al, 1997; Ekam, 2001;
Obochi, 2006).
Coconut water is essentially composed of water,
amino acids, (Arginine, glutamic acid, Aspartic acid, alanine) vitamins
(ascorbic acid, folic acid and pyridoxine), minerals (potassium, nitrogen,
calcium, phosphorus, iron, sodium, chloride, bicarbonate), sugars, fats and
nitrogenous substances (Baptist, 1956; Pandalai, 1958; Suresh and Hedge, 1971;
Prosavior and Rubico, 1979; Davis, 1982; Pehowich et al 2000; Effiong,
2003). The mineral composition of coconut water potentiates it as an oral fluid
for rehydration therapy, and as an antacid, a therapy for peptic ulcer, caused
by high secretion of gastric acid (Church, 1972; Wright and Prescolt, 1973;
Prosaviour et al, 1979; Anzaldo et al, 1980; Davis, 1982; Pehowich et
al, 2000; Effiong, 2003).
Coconut water
has been found to contain hydrolytic enzymes such as cellulase, which breaks
down the wall of kernel to liberate oil, upon which lipase acts to release free
fatty acids and glycerol. Other enzymes present include proteinases which give
rise to various amino acids and nitrogen by hydrolysis of protein in the kernel
(Baptist, 1956; Church, 1972; Anzaldo et al, 1980; Pehowich et al,
2000; Effiong, 2003).
Coconut milk
is essentially composed of high amount of protein, amino acids (glutamine,
Arginine, lysine, leucine, proline), water, sugars (lactose), fats, vitamins
(Ascorbic acid. Nicotinic acid, Biotin pantothenic acid), minerals (nitrogen,
calcium, iron, phosphorus) (Anzaldo et al, 1980; Davis, 1982; Pehowich et
al, 2000; Effiong, 2003).
This current
study focused on the assessment of hepatocellular drug-metabolizing enzyme
induction or inhibition in animals exposed daily to caffeine, caffeine and
coconut water, caffeine and coconut milk, or a combination of the two. Enzyme
activities were examined in whole homogenate (WH) and post-mitochondrial
supernatants (PMS) prepared from the liver of rats within each treatment
regimen.
Materials and Methods
Experimental Animals:
Twenty (20)
wistar albino rats weighing between 150-300g obtained from the disease free
stock of the animal house, Department of Biochemistry, College of Medical Sciences, University of Calabar, Nigeria, were used for the study. The animals were
randomly assigned on the basis of average body weight and litter origin into
four (4) study groups of five (5) animals per group. Each rat in a study group
was individually housed in a stainless cage with plastic bottom grid and a wire
screen top. The animals room was adequately ventilated, and kept at room
temperature and relative humidity of 29 + 20c and 40 70%
respectively with a 12 hour natural light dark cycle.
Treatment Regimen:
The animals
were fed ad libitum with water and rat chow (livestock feeds Ltd,
Calabar, Nigeria). Good hygiene was maintained by constant cleaning and removal
of faeces and spilled feed from cages daily. The control group (1) received via
oral route a placebo (4.0ml of distilled water). Groups 2 to 4 were treated
for a 14day period with 50mg/kg body weight of caffeine, 50mg/kg body weight
of caffeine and 50mg/kg body weight of coconut water, and 50mg/kg body weight
of caffeine and 50mg/kg body weight of coconut milk in 4.0ml of the vehicle via
gastric intubation (i.e, orally using orogastric tubes and syringes)
respectively. The experiments were conducted between the hours of 9.00am and
10.00am daily.
Sample Preparation:
One day after
the final exposure, the animals were anesthetized by inhalation of an over dose
of chloroform, the blood of each rat was collected by cardiac puncture and
centrifuged at 4000 x g for 30 minutes into serum. The liver of each rat was
harvested, ground using mortar and pistle, and buffered with TRIS buffer, pH
7.4. A whole homogenate (WH) was prepared by centrifugation using a centrifuge
at 4000xg for 30 minutes. Subsequently, aliquots of the whole homogenate (WH)
obtained from each rat liver was subfractionated as follows an aliquot of 10ml
of each whole homogenate was placed in a pyrex graduated test tube and was
quantitatively transferred into centrifuge tubes. The tubes were balanced in a
centrifuge and subfractionated by spinning for 10 minutes at 6000xg (1,900rpm).
The resultant 6000xg post nuclear supernatant (PNS) was centrifuged at 10,000xg
for 15 minutes to obtain the post mitochondrial supernatant (PMS), which was
made up to 100ml mark with the TRIS buffer, pH 7.4, in a volumetric flask. The
serum, whole homogenate and the post mitochondrial supernatant were stored at
-700c freezer and used for the various assays.
Biochemical Assays:
The whole homogenate (WH) and post mitochondrial
supernatant (PMS) were used for the analysis of the protein, RNA, and
Protein/RNA ratio; the serum that was collected was used for the estimation of
alanine and aspartate amino transferase (ALT and AST, respectively) activities.
Liver protein was determined using the Biuret method described by Lowry et al
(1951). Liver RNA was determined with modifications of the method of Burton
(1956). Protein/RNA ratio was calculated. Alanine and aspartate amino
transferase (ALT and AST) were determined using the standard methods described
by Mathieu et al (1982).
Preparation of Caffeine:
Synthetic caffeine was obtained from May and Baker
(M&B) limited, Enfield, Middle Sex, United Kingdom (UK), and used for the
study. A stock solution of caffeine was prepared by dissolving 20g of powdered
caffeine in 500ml of hot distilled water. The solution was allowed to cool to
room temperature. Out of the stock solution, 50mg/kg body weight of caffeine
was administered to all the test groups (i.e, groups 2 to 4) in 4.0ml of the
vehicle via gastric incubation respectively.
Preparation of Coconut Milk
and Coconut Water:
A matured
coconut fruit was obtained from a coconut plantation in Calabar, Nigeria, and
used for the study. The fruit was shelled, the nut removed and the water
collected. The nut was then grated using a stainless grater. A stock solution
of coconut milk was obtained by dissolving 50g of the grated mass of coconut in
500ml of distilled water, heated to 650c and the residues removed
using a sieve. The solution was boiled for 30 minutes, and oil scooped. The
solution was again boiled with constant stirring for another 30 minutes, and
the crude milk was allowed to cool to room temperature. 50mg/kg body weight of
coconut water and 50mg/kg body weight of coconut milk obtained were
administered to the animals in groups 3 and 4 respectively in 4.0ml of the
vehicle via gastric incubation.
Statistical Analysis:
Data collected
were expressed as mean + standard deviation (SD) and the student t
test were used for analysis. Values of p<0.05 were regarded as significant.
Results
The results of the administration of caffeine,
caffeine and coconut water and caffeine and coconut milk on levels of hepatic
protein, RNA, protein/RNA ratios, ALT and AST are presented in Tables 1,2,3,
and 4 respectively. The results for caffeine alone (i.e, groups 2) showed that
there was a significant increase (p<0.05) in the values of hepatic protein
contents, as well as in serum ALT and AST activities, in the caffeine-treated
groups when compared to values observed with control rat samples. This finding
was attributable to the increases in protein levels alone, as there were no
significant differences (p<0.05) in values of the hepatic RNA levels and
protein/RNA ratios as compared to those in the controls. However, the results
for the caffeine and coconut water treated groups (ie, groups 3) showed that
there was a significant increase (p<0.05) in the values of hepatic protein
and protein/RNA ratios when compared to those of the controls while there was a
significant decrease (p<0.05) in the values of ALT and AST activities. Also,
the results for the caffeine and coconut milk treated groups (i.e., groups 4)
showed that there was a significant increase (p<0.05) in the values of
hepatic protein contents and protein/RNA ratios when compared to those of the
controls while there was a significant decrease (p<0.05) in the values of
ALT and AST activities.
Comparing the values of the
hepatic protein levels and protein/RNA ratios obtained in the caffeine and
coconut water treated groups to those in the caffeine and coconut milk treated
groups, it was observed that the values of the hepatic protein levels and
protein/RNA ratios in the caffeine and coconut milk treated groups were higher
than those of the caffeine and coconut water treated groups and vice versa for
the values of ALT and AST activities.
However, a
study on the interactions of the three substrates (ie, caffeine, coconut waster
and coconut milk) may be rewarding to diagnosis and or treatment.
Table 1. Effect of treatment
on liver protein content in Wistar albino rats.
|
Group
(N)
|
WH
(mg/ml)
|
PMS
(mg/ml)
|
1. |
Control |
7.12 ± 0.57 |
5.64 ± 0.51 |
2. |
Caffeine |
10.43 ± 0.48* |
8.36 ± 0.43* |
3. |
Caffeine + Coconut Water |
18.66 ± 0.83* |
16.59 ± 0.52* |
4. |
Caffeine + Coconut Milk |
22.68 ± 0.76* |
20.68 ± 0.67* |
N = Number of rats per group = 5. Values are expressed as
mean ± SD. WH = Whole homogenate and
PMS = Post mitochondrial supernatant. * Significantly different from control,
P<0.05, using student t test.
Table 2: Effect of treatment on RNA content in Wistar albino rats
|
Group (N) |
WH (mg/ml) |
PMS (mg/ml) |
1 |
Control |
6.18 + 0.48 |
5.13 + 0.23 |
2 |
Caffeine |
6.29 + 0.57* |
5.48 + 0.34* |
3 |
Caffeine + Coconut Water |
6.67 + 0.58* |
5.63 + 0.48* |
4 |
Caffeine + Coconut Milk |
6.85 + 0.62* |
5.74 + 0.52 |
N = Number of rats per group = 5. Values are expressed as
mean ± SD. WH = Whole homogenate and
PMS = Post mitochondrial supernatant. * Significantly different from control,
P<0.05, using student t test.
Table 3: Effect of treatment on Protein/RNA ratio in Wistar albino rats
|
Group (N) |
WH |
PMS |
1 |
Control |
1.15 + 1.19 |
1.10 + 2.22 |
2 |
Caffeine |
1.67 + 0.84* |
1.53 + 1.26* |
3 |
Caffeine + Coconut Water |
2.80 + 1.43* |
2.95 + 1.08* |
4 |
Caffeine + Coconut Milk |
3.31 + 1.23* |
3.60 + 1.29* |
N = Number of rats per group =
5. Values are expressed as mean ±
SD. WH = Whole homogenate and PMS = Post mitochondrial supernatant. *
Significantly different from control, P<0.05, using student t test.
Table 4: Effect of treatment on enzymes activities in wistar albino rats
|
Group (N) |
ALT (u/l) |
AST (u/l) |
1 |
Control |
22.63 + 0.39 |
37.53 + 0.61 |
2 |
Caffeine |
37.24 + 0.37* |
45.41 + 0.53* |
3 |
Caffeine + Coconut Water |
16.78 + 0.41* |
30.84 + 0.67* |
4 |
Caffeine + Coconut Milk |
10.94 + 0.38* |
20.27 + 0.46* |
N = Number of rats per group = 5. Values are expressed as
mean ± SD. WH = Whole homogenate and
PMS = Post mitochondrial supernatant. * Significantly different from control,
P<0.05, using student t test.
Discussion
In this study,
caffeine independently produced an increase in the activity of alanine and
aspartate amino transferases (ALT and AST), resulting in hepatotoxicity due to
inflammation of the cytoplasm, and a resultant leakage of cytoplasmic enzymes
into the blood stream. These enzymes acted to block the transcription and
translation steps of the genetic code, resulting in decreased processes of
protein biosynthesis. High concentrations of tannic acid inhibited caffeine
uptake, resulting in accumulation of methylxanthines, alanine, aspartate and
nitrogen levels, leading to increased permeability of the liver cells. These
effects, in turn, had led to inhibition of microsomal drug-metabolizing
enzymes. However, an apparent caffeine coconut water and caffeine-coconut
milk interactions decreased the activities of ALT and AST, resulting in
increase in Protein biosynthesis, hepatic protein levels and protein/RNA
ratios. While the increase in the hepatic protein levels and protein/RNA ratios
could be attributed to increased protein biosynthesis (since the RNA content is
negligably affected), the decrease in ALT and AST activities could be
attributed to the decreased permeability of the liver cells arising from
increased glucuronidation (addition of highly polar groups such as sulfates and
glucuronic acid). These effects, in turn, decreased the tannic acid, alanine,
aspartate and nitrogen concentrations in the liver cells, which increased the
rate of gastrointestinal absorption of caffeine metabolites. Thus, it seems
that the decrease in ALT and AST activities enhanced the induction of
microsomal drug-metabolizing enzymes, and these regulatory controls may act to
conserve nutrients and energy for the cells. These results are in consonance
with the studies of Orrhenuis et al (1965), Marshall (1978), Remmer and
Merker (1993), and Singh et al (1998). The reports of these workers
showed that increased protein synthesis due to increased glucuronidation of the
endoplasmic reticulum and decreased permeability of the liver cells resulted in
induction of the microsomal drug-metabolizing enzymes. Also, it is likely that
the toxic effects of caffeine may be modified by the rapid production of
caffeine metabolites in the present of coconut water and cocoanut milk which
could generate reactive electrophiles that could activate microsomal
drug-metabolizing enzymes such as monoxygenase, cytochrome P450,
glucuronyl transferase and nitroreductase (Nerbert and Gelboin, 1970, Matsunura
and Omura, 1973, Bertollni et al, 1980, Salem et al, 1981, Singh et
al, 1998). Also, the vitamins and amino acids present in the coconut water and
coconut milk may likely have a role to play in providing cofactors for the
activation of the drug-metabolizing enzymes and maintenance of liver cell
integrity, even in the presence of hepatotoxic agents like caffeine (Ekam,
2001).
ALT catalyzes
a reversible amino group transfer reaction in the Krebs (tricarboxylic acid)
cycle necessary for tissue energy production while AST catalyzes transfer of
the nitrogenous portion of an amino acid to an amino acid residue. ALT is
released from the hepatocellular cytoplasm into the blood stream when there is
acute hepatocellular damage; AST is found in the cytoplasm and mitochondria of
many cells such as liver, heart and is released into serum in proportion to
cellular congestion due to heart failure (Rodwell, 1996).
The
Protein/RNA ratios are suggestive of enzyme induction and serum endoplasmic
reticulum proliferation (Marshall, 1978; Hunter and chasseaud, 1978; Bertolini et
al, 1980; Salem et al, 1981; Bahtnager and Misra, 1988; Singh et
al, 1998). This is because microsomal enzyme induction could most likely
depends on de novo protein biosynthesis (Bertolini et al, 1980; Rodwell,
1996; Coon, 1996; Singh et al, 1998). In explaining the mechanism of de
novo synthesis, the inducing agent acts to relieve repression of enzyme
synthesis (Rodwell, 1996). The increased synthesis of enzyme protein could also
be as a result of an increase in the translation process due to a reduced rate
of turnover of messenger and other types of RNA (Lessard and Peska, 1971;
Rodwell, 1996; Singh et al, 1998).
The mechanisms of action of
induction of the microsomal drug-metabolizing enzymes therefore, may involve
activation of the transcription process. Transcription is mediated and
regulated by regulatory protein- DNA interactions. The regulatory proteins are
DNA-binding, proteins that recognize specific DNA sequences. Biosynthesis of
these regulatory proteins enhanced glucuronidation of the endoplasmic
reticulum, leading to increased cytoplasmic volume, and increased activities of
the microsomal drug-metabolizing enzymes. Coconut products (ie coconut water
and coconut milk) however, acted synergistically with caffeine to increase the
induction of the microsomal drug-metablizing enzymes (since inducible enzymes
are those whose rate of synthesis, and hence their amount, increases with the
introduction of a specific substrate), leading to a faster clearance and
elimination of caffeine from the body.
The
endoplasmic reticulum (ER) is one of the major sites for protein synthesis in
the cell (Craft et al, 1979; Remmer and Merker, 1993; Balogun and
Malomo, 1998: Ebong et al, 1998). The majority of membrane and secretory
proteins are synthesized on membrane bound ribosomes and are translocated in
the ER where folding, glycosylation, and disulfide bond formation take place to
give the unique configuration and function of the proteins (Remmer and Merker,
1993; Coon, 1996; Raisfield et al, 2000). This may suggest that
synthesis of newenzymes are dependent upon the formation of new functional
membranes, and increased glucuronidation of the endoplasmic reticulum. The
coconut products appeared to have enhanced formation of new functional
membranes and increased glucuronidation of the endoplasmic reticulum, leading
to induction of the microsomal drug-metabolizing enzymes.
In conclusion,
the results have shown that coconut products-caffaine interactions increased
hepatic protein levels and protein/RNA ratios and decreased ALT and AST
activities. These effects in turn, enhanced induction of microsomal drug-metabolizing
enzymes. Thus, the findings of this study may suggest that coconut water and
coconut milk may be used as inducers of microsomal drug-metabolizing enzymes
and or as antidotes to drugs such as alcohol, caffeine and antibiotics.
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