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Biokemistri
Nigerian Society for Experimental Biology
ISSN: 0795-8080
Vol. 18, Num. 1, 2006, pp. 31-37
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Biokemistri, Vol. 18, No. 1, June, 2006, pp. 31-37
Vitamin b12
supplementation: effects on some biochemical and haematological indices of rats
on phenytoin administration
Itemobong S. EKAIDEMa*, MondayI.
AKPANABIATUb, Friday E. UBOHb and OffiongU. EKAb
aDepartment of Chemical Pathology, College of
Health Sciences, University of Uyo, P.M.B. 1017, Uyo, Nigeria
bDepartment of Biochemistry, University of
Calabar, Calabar, Nigeria
*To whom correspondence should be addressed. Email:seityjen@yahoo.com, Tel: 0802-3884258
Received June 9, 2005
Code Number: bk06006
Abstract
Phenytoin
is known to have some toxicological implications. Vitamin B12
supplementation during phenytoin administration was investigated to assess the
benefits and risks of single vitamin supplementation. This study evaluated the
biochemical and haematological effects of vitamin B12 on phenytoin
toxicity. Twenty-four experimental animals were divided into 3 groups of 8
rats each. The control (group 1) received distilled water as placebo. Groups
2 and 3 were given 5mg/kg body weight of phenytoin for 4 weeks while group 3 in
addition to phenytoin received intra-peritoneal administration of 15μg/kg
body of vitamin B12 twice a week. Biochemical parameters such as
AST, ALT, ALP, lipid profile and haematological indices were assayed as indices
of toxicity. The result of the study showed that phenytoin administration
resulted in anaemia which was ameliorated by vitamin B12
co-administration. Phenytoin also increased significantly the leukocyte count
upon which B12 had no effect. Liver enzymes activities were
significantly (p<0.05) raised during phenytoin administration and
interestingly B12 further increased the level of these enzymes.
Administration of phenytoin only gave a significant (p<0.05) increase in the
level of serum Low density lipoprotein cholesterol. Serum cholesterol, TG and
HDL-chol were not significantly affected. Although there was no significant
change in serum cholesterol, the slight increase was more than 1% which is
capable of causing a 3% increase in the risk of coronary heart disease. A
significant decrease was also noted when phenytoin was supplemented with B12.
We observed that vitamin B12 co-administration is beneficial in
remitting anaemia and the atherosclerotic risk caused by phenytoin but may
enhance hepatotoxicity. By this result we would therefore suggest that the use
of vitamin B12 alone as supplement during phenytoin administration
be discouraged.
Key
words: Phenytoin, serum enzymes,
lipid profile, vitamin B12, atherosclerosis, hepatotoxicity.
INTRODUCTION
Vitamin
supplementation is known to impart significant benefits in terms of disease
prevention and treatments and has been widely accepted as a measure of control
of micro nutrient deficiencies. Vitamin B12, generally called
cobalamin, is a porphyrin like ring compound with central cobalt atom attached
to a nucleotide. Its anti-anaemic function has been known for years. Recent
studies have shown that appropriate amount of vitamin B12 can
protect against dementia1,2, boost immune function and maintain the
nervous system3. Also, vitamin B12 lowers homocysteine
levels and protects against atherosclerosis and other cardiovascular disease4,5,
as well as certain neurological diseases associated with increased homocysteine
including Alzheirmers diseases 6,7; depression and schizophrenia8.
Vitamin B12 also plays a vital role in maintaining methylation
reactions that repair DNA and hence prevents cancer9,10.
On
the other hand, vitamin supplementation during some specific drug therapy in
diseases may be deleterious to health and hence defeat the very purpose of its
administration11. These effects may be due to drug nutrient
interactions resulting in either alteration in absorption and metabolism of the
vitamin or increased metabolic clearance of the drug hence compromised therapeutic
benefit. A number of drugs are known to reduce the absorption of vitamin B12
in the gastrointestinal tract. These include proton-pump inhibitors (e. g.
omeprazole, lansoprazole) and H2- receptor antagonists (e. g.
Tagamet, pepsid, zanatac). These drugs markedly decrease stomach acid
secretion required to release dietary vitamin B12 from foods, thus
long-term use of proton-pump inhibitors were associated with decreased level of
vitamin B12 in the blood12; however, long-term use of H2-
receptor antagonists did not result in vitamin B12 deficiency since
the action of the drug is not always prolonged13. Metformin
decreases vitamin B12 absorption by tying up free calcium required
for absorption of the IF-B12 complex 14. Other drugs
which inhibit vitamin B12 absorption are cholestyramine,
chloramphenicol, neomycin and colchicines. Nitrous oxide inhibits the two
vitamin B12 dependent enzymes and hence can produce many of the
clinical features of vitamine B12 deficiency15.
Phenytoin
is a hydantoin anticonvulsant used widely in the management of generalized and
partial seizures16,17. It has been shown to alter the
bioavailability and metabolism of vitamin B12 and folic acid18,19.
Over 50% of patients on long-term phenytoin therapy demonstrated low serum and
red cell levels of vitamin B12 and folic acid20-22.
Several suggestions have been made in attempts to explain the mechanism by
which phenytoin alters the metabolism of folates18,20,23, however,
reports on phenytoin-vitamin B12 interaction are quite scanty. Since
some of the actions of vitamin B12 are dependent on folate cofactor,
one may suggest that the altered level of B12 by phenytoin is
related to the phenytoin induced folate deficiency. This argument is based on
the observation that administration of folic acid resulted in elevated serum
level of vitamin B12 in epileptic patients22.
The
complication of phenytoin therapy has called for serious concern since the drug
is capable of disrupting tissue integrity24,25. Anaemia and
hepatotoxicity are common findings during phenytoin therapy17,26.
The heapatotoxicity of phenytoin was accompanied by rashes, fever,
lymphadenopathy and eosinophilia, which suggest that the mechanism of toxicity
may be a hypersensitive reaction27, 28. The phenytoin hypersensitivity
syndrome is not fully understood. However, phenytoin metabolism which is
mediated by a group of mixed-function oxidases known as cytochrome P450
and the intermediate metabolites, known as arene oxides, are important to the
immunological responses. The cytotoxic activity of these oxides has been
reported and epoxide hydroxylase are responsible for their detoxification26.
Indivuals that developed phenytoin hypersensitivity syndrome are reported to
have lost the ability to detoxify arene oxides and it is believed that family
members may have similar inability to metabolize arene oxides, thereby
confirming the report in familial cases29. Folate is hypothesized
to be a cofactor in phenytoin metabolism and may be responsible for the changes
in pharmacokinetics of phenytoin usually leading to lower serum phenytoin
concentration and seizure breakthrough in patients taking folate
supplementation20. The involvement of vitamin B12 in the
metabolism of phenytoin and a possible modulatory role of vitamin B12
on phenytoin toxicity in patients have not been established.
The
use of vitamin supplement in both health and diseases; especially in areas,
such as Nigeria, where locally sourced daily diets are deficient in essential
vitamins; is highly encouraged in view of its health benefits. However, the
health risk of individual vitamin used alone and in combination with others in
certain disease conditions where drug therapy is instituted is sometimes
overlooked. Against this background, we examined the effects of vitamin B12
supplement on phenytoin toxicity using experimental rats.
MATERIALS AND METHODS
Animals
Twenty-four growing albino wistar rats weighing 120g
to 160g were obtained from the Department of Biochemistry, University of Calabar, Calabar, Nigeria. The
animals were kept in a well-ventilated room of standard laboratory condition.
They were fed with normal rat formula (Pfizer livestock Co. Ltd. Aba,
Nigeria). All the animals were randomly divided into three groups of eight
rats each. The control (group 1) received distilled water as placebo. Group 2
and 3 were treated with phenytoin and group 3 in addition to phenytoin received
vitamin B12.
Administration
of phenytoin and vitamin b12
Commercially
available phenytoin capsules were obtained form Parke-Davis, Hoofirea. Vitamin
B12 vials were obtained from Vitabiotic (Nigeria) Ltd.
Lagos, Nigeria. 5mg/kg body weight of phenytoin was administered orally to
all rats in group 2 and 3; while 15μg/kg of cyanocobalamin (vitamin B12)
was given intraperitoneally twice a week to rats in group 3 in addition to
phenytoin. The treatment lasted for four weeks.
The
animals were sacrificed by suffocation in chloroform fumes and blood collected
by cardiac puncture. The blood samples were divided into two. One part was
collected into EDTA tubes for haemoglobin, PCV and white blood cell count
determinations; while the other part was collected into plain tubes and allowed
to clot and retract at room temperature for two hours. Sera were separated by
centrifugation at 3000g for 5min using bench top centrifuge (MSE minor England). The
sera were collected into sterile plain tubes and stored in refrigerators for
analysis. Haemoglobin determinations were performed immediately while all
analyses on serum were completed within 24 hours of sample collection.
Heamatocrit
Determination
Haematocrit
was estimated by using the method of Alexander and Griffiths30.
Haematocrit tubes were filled by capillary action to the mark with whole blood
and bottom end of the tubes were sealed with plasticide and centrifuged for 4
minutes using haematocrit centrifuge. The percentage cell volume was read by
sliding the tube along a critocap chart until the meniscus of the plasma
intersects the 100% line.
Haemoglobin
Determination
Cyanmethaemoglobin (Drabkin) method of haemoglobin
estimation31 was employed. 0.02ml of anticoagulated whole blood
sample was added to 5ml of Drabkin reagent, mixed and incubated for 5 minutes
at room temperature for the colour to develop and the absorbance was read
against reagent blank at 540nm using DR 3000 spectrophotometer.
Total
White Blood Cell Count
The
estimation of total white blood cell counts was done by visual means using New
Improved Neubauer counting chamber32. A 1 in 200 dilution of blood
was made in Turks fluid and the counting chamber with its cover glass already
in position was immediately filled with the diluted blood using a Pasteur
pipette and ensuring that the chamber was filled in one action. The charged
chamber was allowed to remain undisturbed for 2 minutes to allow the cells to
settle. The cells were them counted using x40 eye piece. Four squares at the
corners of the chamber were counted.
Biochemical
Determination
Hepatic
enzymes of clinical significance and serum lipid profile were determined in
samples spectrophotometrically using kit methods. Serum aspartate
aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase
(ALP) activities were measured using Randox kits. Serum total protein was determined
with biurete kit method (Randox U. K.). Total cholesterol, Triglycerides (TG)
and HDL cholesterol were also measured using Randox kits. The absorbances of
all the tests were determined using spectrophotometer (HAICH, DR 3000, Germany). LDL
cholesterol was obtained by calculation using appropriate relationship
between total cholesterol, TG and HDL cholesterol33.
Statistical
analysis was carried out using students t-test. A probability of 0.05 was used
as a level of significance.
RESULTS
The effects of vitamin B12 supplementation
during phenytoin administration in rats were investigated to assess the
benefits and risk of single vitamin supplementation. Percentage weight gain,
haemoglobin level, haematocrit, total white blood cell count, total serum
protein, liver function enzyme activities and serum lipid profile were measured
as indices of phenytoin toxicity. Table 1 shows the effects of vitamin B12
on haematological indices, weight gain and total serum protein. Weight gain
(%) in group 2 (24.46 ± 4.32) and group 3 (18.76 ± 4.03) were significantly
(p<0.05) lower than that of control group (37.67 ± 6.89). Haemoglobin
(g/dl: 7.55 ± 0.68) and haematocrit (%: 38. 64 ± 4.37) were significantly
decreased (p<0.05) by phenytoin treatment; but supplementation vitamin B12
(group 3) raised their levels to control values. Total white blood cell count
was significantly increase while total serum protein was decreased (p<0.05)
in group 2 treated with phenytoin only; these changes were, however, not affected
by vitamin B12 supplement. The effects of vitamin B12 on
liver function enzymes activities and lipid profile is shown in table 11.
Serum AST and ALP of phenytoin treated rats (group 2: 54.13 ± 4.91 and 151.90 ±
30.33 respectively) increased significantly (p<0.05) when compared with
control (37.88 ± 9.39 and 118.61 ± 20.90 respectively); while ALT (26.75 ±
3.41) increased marginally, supplementation with vitamin B12 further
increased the activities of the three enzymes (AST: 73.17 ± 5.44; ALT: 36.38 ±
6.35; ALP: 192.66± 31.65) when compared with group 2 and with control. Total
cholesterol was marginally increased while LDL- cholesterol showed
significantly increase (p<0.05) in group 2 rats when compared to control.
The levels of TG and HDL cholesterol were not significantly affected.
Vitamin B12 supplement was beneficial as it lowers the levels of
cholesterol, TG and LDL-cholesterol to normal values.
Table 1: Effect
of vitamin b12 on haematological indices, weight gain and total
protein
GROUPS
|
TOTAL PROTEIN (g/100ml)
|
Weight Gain (%)
|
PCV (%)
|
Hb
(g/100ml)
|
WBC x103/ml
|
CONTROL(gp1)
|
8.71±0.58
|
37.67±6.89
|
48.63±2.62
|
10.11±0.84
|
4.60±1.07
|
PHENYTOIN ONLY (gp 2)
|
6.71±0.87*
|
24.64±4.32*
|
38.64±3.7*
|
6.85±0.68*
|
6.14±0.96*
|
PENYTOIN + VITAMIN B12 (gp3)
|
6.46±0.91*
|
18.76±4.03**
|
49.15±2.90*
|
10.13±0.86
|
6.44±1.08*
|
Values are expressed as mean ± SD, n = 8: *p<0.05,
**p<0.01
Table
2: Effect of vitamin b12
on liver enzymes and lipid profile
GROUP
|
AST
(iu/1)
|
ALT
(iu/1)
|
ALP
(iu/1)
|
TCHOL
(mmol/1)
|
TG
(mmol/1)
|
LDL-
Cho (mmol/1)
|
HDL-Chol
(mmol/1)
|
CONTROL
(gp1)
|
37.88±9.39
|
23.63±5.26
|
118.61±20.90
|
2.12±0.22
|
0.75±0.22
|
0.21±0.21
|
1.57±0.19
|
PHENYTOIN
ONLY (gp2)
|
54.13±4.91*
|
26.75±3.41
|
151.90±30.33*
|
2.73±0.43
|
0.92±0.29
|
0.82±0.23**
|
1.49±0.13
|
PHENYTOIN
+ VITAMIN B12 (gp 3)
|
73.17±5.44**
|
36.38±6.35*
|
192.66±31.65**
|
2.23±0.48
|
1.06±0.31
|
0.22±0.18
|
1.53±0.14
|
Values
are expressed as mean ± SD, n = 8: *p<0.05, **p<0.01
The
results showed that although vitamin B12 supplement is beneficial by
remitting anaemia and reducing the atherosclerotic risk in phenytoin treated
rats, the hepatotoxic effect of phenytoin may be enhanced.
DISCUSSION
In this study, growth retardation, increased risk of
cardiovascular diseases, anaemia, and hepatocellular toxicity were observed in
young albino wistar rats treated with therapeutic dose of phenytoin. A
significant reduction in percentage weight gain was associated with phenytoin
treatment and even in combination with vitamin B12. This finding
that vitamin B12 supplement could not cause significant weight gain
in rats receiving phenytoin treatment suggests the importance of the presence
of other vitamins including folic acid for proper growth of the animals.
Reports have shown that phenytoin administration causes alterations in the
metabolism of vitamin B12 and folic acid20,23 which are
essential in DNA synthesis and cell proliferation.
The
significant difference in the value of total serum protein, observed in this
study, indicates that liver cell integrity may have been affected by the
treatment. Since hepatocytes constitute a major source of serum protein,
decreased protein level may therefore result from the effect of the drug on
liver cells resulting in decreased protein synthesis. The phenytoin-induced
anaemia was remitted by vitamin B12 treatment as evident by raised
haemoglobin and PCV levels in group 3 rats. The activities of AST, ALT and ALP
were increased by phenytoin treatment in rats. These findings agree with
previous report that long-term therapy with phenytoin caused increased AST,
ALT, ALP and enlargement of the liver in epileptic patients34. The
increased activities of liver function enzymes in this study indicate possible
hepatocellular damage. Also the decrease in total serum protein and the
increased circulation white blood cell further points to impaired liver
function and cellular inflammation.
Interestingly,
vitamin B12 supplementation during phenyton treatment resulted in
further increases in the activities of liver functions enzymes; AST, ALT and
ALP, but the levels of serum protein and circulating white blood cells in these
rats were not significantly different from those without vitamin B12
supplement. These findings showed that vitamin B12 supplement does
not remit the hepatotoxicity of phenytoin but may rather enhance it and that
vitamin B12 may not have a significant role in the metabolic
clearance of phenytoin. Phenytoin is reported to lower the serum and red cell
levels of vitamin B12 and folate21,35. Phenytoin-induced
folate depletion in rat liver has also been reported23. Thus, the
mechanism of phenytoin hepatoxicity may, in addition to phenytoin
hypersensivity syndrome 17,27, be related to the drug-induced
alteration in folic acid-vitamin B12 interactions. The conversion of
coblamin to methylcobalamin requires 5-methyltetrahydrofolates.
Methylcobalamin is an important coenzyme of vitamin B12 which
participates in the convertion of homocysteine to methionine. In addition to
increased folate requirement for metabolic clearance of phenytoin and
phenytoin-induced folate malabsorption, the conversion of cobalmin to
methylcobalamin may constitute an additional constrain to the availability of
folate for other cellular functions. This may be the reason for enhanced
phenytion hepatotoxicity by vitamin B12 co-administration without
additional source of folic acid.
Studies
on serum lipid profile showed only marginal increases in total cholesterol and
a significant increase in LDL cholesterol in rats treated with phenytoin
without vitamin B12 supplement. These findings are in line with
report of Luoma et al36 who demonstrated increases in serum
cholesterol and triglyceride levels in healthy volunteers and epileptic
patients treated with phenytoin. Our studies have shown that phenytoin-induced
increases in total cholesterol, LDL-cholesterol and hence the atherosclerotic
risk could be reduced by administration
vitamin B12. The effect of vitamin B12 supplementation
on lipid kinetic during phenytoin
treatment may not be unconnected with the action of 5-deoxyadenosylcobalamin, a
coenzyme of L-methylmalonylCoA mustase
which catalyze the conversion of L-methylmalonyl-CoA to succinyl-CoA, an
important reaction in energy production from fats and proteins.
In
conclusion, we observed that vitamin B12 co-administration during
phenytoin therapy remits anaemia and the atherosclerotic risk caused by
phenytoin but may enhance its hepatotoxicity. Therefore we suggest that the
use of only vitamin B12 as a monovitamin supplement during phenytoin
administration be discouraged.
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© 2006 Nigerian Society for Experimental Biology.
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