The effects of bile salts on the kinetic properties of enzymes used in the diagnosis of liver and kidney damage.

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-diphenyl tetramlium (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