Journal of APPLIED
BIOMEDICINEISSN 1214-0287 (on-line)
ISSN 1214-021X
(printed)
Volume 5 (2007)
Lactate dehydrogenase isoenzyme pattern in the liver tissue of
chemically-injured rats treated by combinations of diphenyl dimethyl
bicarboxylate Laila Faddah, Nabil Abdel- Hamid, Yaser Abul -Naga,
Sanaa Ibrahim, Ahmed Mahmoud Address: Nabil Mohie Abdel-Hamid,
Department of Biochemistry, Faculty of Pharmacy, El-Minia University, El Minia,
Egypt
nabilmohie@yahoo.comFull text article
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Received 15th October 2006.
Revised 22nd
December 2006.
Published online 12th March 2007.
Summary
The purpose of our study is to evaluate the protective effect of diphenyl dimethyl
bicarboxylate (DDB) in combination with some antioxidants, namely vitamin C
(V.C), vitamin E (V.E), and selenium (Se), in liver damage induced by carbon
tetrachloride (0.2 ml / Kg body weight). This was done by monitoring the liver
total and fractional lactate dehydrogenase ( LDH ) activities. The results
revealed a significant increase in the activity of liver total LDH activity in
CCl4 – intoxicated rats with a significant increase in both LDH3 and 4 and a
significant decrease in LDH5. LDH2 disappeared after CCl4 treatment and neither
DDB nor its combinations could restore this permanent change. DDB alone
significantly decreased the CCl4 - raised total LDH and LDH4, but still far from
the control and failed to correct LDH3 and 5 variations. A combination of DDB
and V.C, V.E and Se showed the best corrective potential in both total LDH and
LDH3 activities, without correcting the increased LDH4, nor the decreased LDH5
isoenzyme. Although this combination was previously reported to correct liver
function disturbances, it seems that CCl4 and consequently hepatitis C may
induce some irreversible, non-curable changes by DDB or even by additional
antioxidants. Its clinical usefulness seems to be through different metabolic
pathways, not including correction of LDH disturbances, which necessitates
additional investigation for other adjunct medicines for treating liver fibrosis
in clinical practice.
Keywords: LDH isoenzymes – liver fibrosis – DDB – CCI4 – rats
INTRODUCTION
DDB has
been used in the treatment of abnormal liver functions without noticeable side
effects (Qing and Liu 1992). It was originally used in China in 1977 in the
treatment of hepatoma and chronic hepatitis. The main pharmacological actions of
DDB are protection of hepatoc-ytes against injury and increase in the
detoxication capacity of the liver, so it reverses the effect of CCl4 on
transaminases in rats (Zhu et al. 1999). LDH is an oxidoreductase enzyme in the
glycolytic pathway whose activity is necessary for the reversible reaction in
which pyruvate and lactate are inter converted (Kory and Susan 1993). Its
isoenzyme patterns are characteristic of many tissues such as: LDH1, LDH2 found
in the heart erythrocytes and kidney; LDH4, LDH5 found in the liver, and LDH3 in
lymphatic tissue, platelets and many malignant tissues (Henderson 1986). Tissue
LDH concentration is much higher than in serum or plasma, so the leakage of LDH
from damaged cells is reflected in the serum LDH pattern. Changes in the serum
LDH pattern have been employed for the detection of physiological changes in
humans (Rodrigue 1995). LDH isoenzyme ratios in relation to total LDH seem to be
species and even organ dependent (Salplachta and Necas 2000).
The purpose of our study is to evaluate the protective effect of DDB in combination with
vitamin C, E and / or selenium in carbon tetrachloride intoxicated rats. This
was done by monitoring total LDH activity and separation of LDH isoenzymes by
electrophoresisin a trial to explore the mechanism of the drug on the liver
tissue level.
MATERIALS AND METHODS
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Drugs and animals
DDB was imported from China, ground into powder, then suspended in water using gum
accacia just prior to administration. CCl4 was obtained from Prolabo, UK. Eighty
four male Wistar albino rats of age ranging from 7–8 weeks and weighing
110–115g, were classified into seven groups; 12 rats each.They were given
standard feed, allowed free access of water, kept at temperature range of 20 –
22 and left in a moderately humid room to acclimatize for ten days before
dosing. Rats were kept in polyethylene cages of 50 cm length, 35 cm width and 30
cm heights, under a controlled 12 hours dark /12 hours light cycle.The first
untreated group served as control and was given saline daily orally. The second
group was given single pure CCl4, 0.2 ml / Kg B.W (Kim et al. 1999). The
remaining five groups were given CC14 as mentioned before and were divided as
follows: The third group was treated with DDB (300 mg / Kg .b .wt.) (Fu and Liu,
1992). The fourth was given DDB + V.C (250 mg / Kg, B.W ), orally (Palmer et al
1998).The fifth group was given DDB + V.E (300 mg / Kg, B.W, orally, daily),
(Liang et al 1995) .The sixth group was given DDB +Se (0.3 mg / Kg ,P.O, daily)
(Berne et al 1991). The seventh group was given DDB + V.C + V.E + Se daily. All
treatments were for one month.
METHODS
Liver and heart tissues were excised immediately after 30 days of treatment. Pending analysis, the
tissues were stored frozen at – 20oC. They were homogenized by a high speed
blinder in a solution of 0.9% NaCl, 5 mmol TRIS – HCl buffer (pH 7.4). Extracts
were collected after centrifugation for 30 minutes at 20oC, 15000 Xg, then LDH
activity was analyzed in the clear supernatant, (Babson and Babson 1973).
Polyacryamide gel was prepared as 5.5% in TRIS – HCl buffer, pH 8.6 (3mm inner
diameter, 5cm length). Liver extracts were electrophortetically analyzed against
heart extracts as a standard control for LDH isoenzymes pattern, (Dietz and
Lubrano 1967). Data were analyzed by one way ANOVA and mean values ± SEM were
calculated using Duncan 's new multiple range test (Duncan 1955) .
RESULTS
CCl4 injection produced a significant increase in total
lactate dehydrogenase activity in liver tissue, inhibited the synthesis of LDH2,
significantly elevated both LDH3 and LDH4 and depressed LDH5, compared to the
control value. Administration of DDB alone significantly decreased total LDH,
and did not change LDH3, 4 and 5 values induced by CCl4. The combination with
vitamin C or E and Se, significantly decreased the total LDH activity induced by
CCl4. It also significantly depressed CCl4 – elevated LDH3 but didn't affect
changes in LDH4 and LDH5 (Table 1).
DISCUSSION
LDH is an enzyme present in all human cells catalyzing the pH dependent interconversion of
lactate into pyruvate. Characteristically, human LDH can be separated into five
different isoenzymes (LDH1 though LDH5), based on their electrophoretic mobility
(Kory and Susan 1993). In the present study, CCl4 was used to induce liver
damage in rats. It significantly elevated hepatic enzyme activity of total LDH,
LHD 3, 4, depressed LDH 5 and abolished LDH2. The effect on LDH was
significantly improved by treatment with DDB, but not recovered to normal.This
effect was reported on the serum level, as DDB in a dose of 300 mg/kg body
weight could alleviate the CCl4 effect on total LDH activity while lower doses
failed to show this protective action (El-Sawy et al 2002). Liver necrosis is
known to be associated with decreased LDH 1, and 2 and increased LDH3 and 4
(Yasuda et al. 1989).
Table 1. Effect of DDB and its combinations with
V.C , V.E and Se on LDH activities and isoenzyme pattern in CCl4 -intoxicated
rats after 30 days of treatment (n=12)
|
Group
|
Total LDH |
LDH1 |
LDH2 |
LDH3 |
LDH4 |
LDH5 |
|
Normal
control |
1.82 ± 0.08 |
- |
1.1± 0.1 |
1.2 ± 0.05 |
1.2 ± 0.05 |
96.5 ± 0.2 |
|
CCl4 -intoxicated |
3.46 ± 0.08 * |
- |
- |
2.2 ± 0.05 * |
55.3 ± 0.8* |
42.5 ± 0.7* |
|
CCl4 + DDB |
2.93 ± 0.08 * |
- |
- |
2.7 ± 0.3 * |
47.8 ± 0.4 * |
49.5 ± 0.5 * |
|
CCl4 + DDB + V.C |
1.9 ± 0.08 * |
- |
- |
2.9 ± 0.06 * |
50.8 ± 0.9 * |
46.3 ± 0.52 * |
|
CCl4 + DDB + V.E |
2.18 ± 0.11 * |
- |
- |
1.98 ± 0.3 * |
56.1 ± 0.4 * |
41.9 ± 0.4 * |
|
CCl4 + DDB + Se |
2.82 ± 0.06 * |
- |
- |
1.5 ± 0.14 * |
56.5 ± 0.5 |
42 ± 0.4 * |
|
CCl4 + DDB + V.C +V. E+ Se |
1.88 ± 0.03 * |
- |
- |
1.65 ± 0.16 * |
49.2 ± 2.6 * |
42.8
±0.4 |
*
Significantly different from Control
Hepatic LDH activity was significantly lower in the DDB treated groups
supplemented with both vitamins and Se when compared to the corresponding CCl4
treated rats. Supplementation with vitamins could be considered as a possible
mechanism for potentiating DDB antioxidant power. Moreover, it was speculated
that it would increase the availability of hepatic levels of both V.C and V.E,
which would explain the non – enzymatic antioxidant potential of the drug (Ip
and Ko 1996).
In our study, the normal control group was found to have four
LDH isoenzymes in liver tissue demonstrable by electrophoresis, as LDH 1 was
absent. The electrophoresis of the LDH isoenzyme in the DDB treated groups
supplemented with the three vitamins and Se showed an improvement in LDH3
without any benefit on both LDH4 and LDH 5.However, LDH2 is still absent after
this treatment. DDB was reported as directly protecting hepatocyte DNA from
fragmentation and oxidative damage due to chemical – induced liver injury. This
was manifested by its potential in reducing the elevated serum alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) activities, total
bilirubin, serum total bile acid production and tumor necrosis factor alpha and
reducing its messenger RNA expression in liver tissue, both in and ex vivo (Gao
et al. 2005).
CONCLUSION
DDB was proved to have an important
hepatoprotective role against CCl4-intoxication through improvement of normal
hepatic indices and the antioxidant properties. The results of our study
revealed that the level of hepatic LDH in the CCl4 intoxicated rat group is
significantly higher than that of normal control, and elevation of isoenzymes
LDH3, LDH4 and depressed LDH5, with the disappearance of isoenzyme LDH2. After
DDB treatment with vitamins and Se, a significant improvement was noticed. Only
total LDH returned near to normal, while LDH isoenzyme still disturbed. So it is
clear that DDB supplemented with the mentioned antioxidants only restored total
LDH activity near to normal without any role on individual isoenzymes . This
mechanism mostly needs further clarification on the sub cellular level to study
the possible role of DDB on transcriptional events before the synthesis of
individual LDH isoenzymes. Moreover, the corrective action of the drug and its
additives doesn’t rely on the LDH pathway.
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