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76s Biochemical Society Transactions (1997) 25
The effects of bile salts on the kinetic properties of enzymes
used in the diagnosis of liver and kidney damage
WINSTON A. MORGAN, BALWANT KALER and PETER H.
BACH
Interdisciplinary Centre for Cell Modulation Studies, University of
East London, Faculty of Science and Health, Romford Road,
London E l 5 4LZ.
The determination of serum and urinary enzymes levels is widely
used to assess both liver and kidney damage. However, during
obstructive liver disease there is a concurrent elevation in the
concentrations of serum and urinary hydrophobic bile salts, which
have been reported to alter the activity of a number of enzymes
making the accurate assessment of enzymic activity difficult [l].
Bile salts are also used in a number of in vitro studies in an attempt
to determine their mechanism of cytotoxicity [2]. However, the
methods often used to assess cytotoxicity rely on the determination
of enzyme activity such as the leakage of lactate dehydrogenase or
the reduction of dimethylthiaml-2-yl)-2,5-diphenyltetramlium
(MTT), consequently the results obtained may be compromised
where bile salts interfere with these enzyme activity. Presently there
is little information on the direct effects of bile salts on the kinetics
of enzymes widely used in clinical diagnosis or in routine
biochemistly. The present study assesses the effect of three
hydrophobic bile salts (chenodeoxycholic acid, deoxycholic acid
and lithocholic) on the kinetic properties of four enzymes (lactate
dehydrogenase (LDH), glutamate dehydrogenase (GDH), alkaline
phosphatase (AP) and y-glutamyltransferase (y-GT)) which are
widely used either in clinical diagnosis or as biochemical markers
of cell injury.
The enzymes were extracted from the kidneys of male Wistar
rats (250-3OOg). Kidneys were cut into small pieces and placed in
ice cold Earle's-Hepes buffer (pH 7.4) then homogenised using a
Potter-Elvehjem teflon-glass homogenizer. The homogenate was
then centrifuged for 20 min at lOOOg and stored at -20°C. Prior to
assay the homogenate was then centrifuged at lOOOg for 20 min and
the supernatant used to determine enzyme activity in the presence
of the bile salts. The enzymes were determined by standard
biochemical methods [3,4]. Kinetic parameters were determined
from either a plot of substrate concentration against initial velocity
or from a Lineweaver-Burk plot.
Bile salts are potent detergents which are thought to be toxic by
a number of mechanisms including the solubilizing of membrane
components, the inhibition of mitochondrial respiration and the
generation of reactive oxygen species [5,6]. In the present study bile
salts appear to be potent inhibitors of enzyme activity, inhibition of
LDH, AP and GDH by the three bile acids appears to be noncompetitive, while the bile salts has a slight activating effect on y
GT. Lithocholic acid the most hydrophobic bile salt used the study
caused the greatest inhibition while chenodeoxycholic and
deoxycholic had similar effects on enzyme activity. The
mitochcndrial enzyme GDH was the most susceptible to the bile
salts; lithocholic (lOOpM), chenodeoxycholic (1000pM) and
deoxycholic acid (lOOOpM) decreased the V, of GDH by 88% (P
< 0.001), 95% (P < 0.001) and 94% (P c 0.001) as shown in (Table
1). Chenodeoxycholic (lOOOpM) and deoxycholic acid (1000pM)
decreased the V, of LDH by 66% and 48% (P < 0.001)
respectively, chenodeoxycholic (lOOOpM) and lithocholic acid
(100pM) decreased Ap activity by 39% (P < 0.001) and 15%
respectively, while the effect of the bile salts on yglutamyltransferase was not significant (Table 1). Lithocholic acid
the most hydrophobic bile salt used in the study caused the greatest
inhibition while chenodeoxycholic and deoxycholic had similar
effects on enzyme activity, this is in agreement with previous
studies which suggest cytotoxicity of bile salts correlate with
hydrophobicity [2]. The mitochondrial enzyme GDH was the most
Table 1. The effect of hvdrophobic bile salts on ennlme kinetics
K, (mM)
GDH
3.77
0.83**
1.7**
1.8
(n=4)
LDH
0.0145
0.0078*
0.013
ND
V, (% control)
GDH
11.6**
5**
6.3**
(n = 4)
Control
Cheno (1000pM)
Deoxy ( IOOOpM)
Litho (100pM)
AP
0.022
0.020
0.020
0.022
Cheno (1000yM)
Deoxy (1000pM)
Litho (lOOpM)
AP
61**
ND
85
LDH
34**
52**
ND
S T
1.52
2.05
1.76
1.3
S T
94
112
107
The enzymes were entracted from the kidneys of male Wistar rats.
Enzyme activity was determined by incubating the enzyme with
increasing substrate concentration. The K, and V, values were then
extrapulated from either a plot of substrate concentration against
initial velocity or from a Lineweaver-Burk plot. * P < 0.05 and **
P < 0.001 significantly different from control.
~
~~
susceptible to the bile salts; and may in part explain the inhibition
of mitochondria1 respiration. The effect on LDH and AP suggest
that bile salts are capable of inhibiting both membrane-bound and
cytosolic enzymes. These results suggest that any assessment of
enzyme activity in presence of bile acids must take into
consideration the direct effect bile acids on the enzyme.
Furthermore, these results suggest that the alteration of enzyme
activity observed at concentrations of bile salts found in this study
may play a role in the liver damage and the renal failure observed
during obstructive jaundice.
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Bergmeyer, H.U. and Bernt, E. (1978) Methods Enzymat. Anal.,
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Plumber, D.T. and Ngaha, E.O. (1978) Nephrotoxicity, pp 175191. N.Y. Masson Inc.
Aoyagi, T. and Lowenstein, L. (1967) Gastroenterology. 4, 686691.
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