Induction of plasma and tissue enzymes by drugs: significance in toxicological studies.

1. Observations of drug metabolism enzyme induction in animals during early toxicological trials, and phase I or II of clinical trials, are of importance for the development of a new chemical of pharmacological interest. 2. Induction can be detected by determination of enzyme activities or enzyme proteins in tissues (subcellular fractions or cells in culture). 3. Indirect methods for checking induction can involve determinations of endogenous (i.e. glucaric acid, 6-beta-hydroxycortisol) or xenobiotic (antipyrine) metabolites which are produced by the inducible enzyme systems. 4. Recent progress in the knowledge of biochemical mechanisms of induction and the large number of identified isoenzymes in each enzyme 'family' have complicated the interpretation in pharmaco-toxicological studies. 5. This paper describes the present state of the art concerning the relationships between the isoenzymes and the major classes of inducers concerning cytochromes P-450, UDP-glucuronosyltransferases, gamma-glutamyltransferase and epoxide hydrolases.

[6 beta-hydroxycortisol: a non-invasive reflection of enzymes of drug metabolism]. / Le 6-beta-hydroxycortisol: un reflet non invasif des enzymes du métabolisme des médicaments.

6-beta-hydroxycortisol (6-beta-OHF) is the main unconjugated metabolite of cortisol in human urine. 6-beta-OHF could be assayed in urine by several methods : chromatography followed by colour reaction, high performance liquid chromatography, radioimmunoassay and enzyme immunoassay. Urinary 6-beta-OHF is increased in some physiological conditions (such as pregnancy) and pathological states (such as hypercortisolemia and liver disease). The measurement of 6-beta-OHF in human urine may provide a useful index of enzyme induction, its excretion being enhanced by many inducers of the mixed function oxygenase.

A new enzyme immunoassay of microsomal rat liver epoxide hydrolase.

Antiserum against purified rat liver microsomal epoxide hydrolase was produced in the rabbit. We developed an enzyme-linked immunosorbent assay which is reliable with regard to its analytical criteria. The concentration of epoxide hydrolase was measured in liver microsomes of control rats and animals treated with F 1379 (250 mg/kg/day) for 5, 7, 14, and 21 days. This hypolipidemic drug was able to induce strong epoxide hydrolase activity and enhance protein concentration. The gradual increase in epoxide hydrolase concentration paralleled the increase of epoxide hydrolase activity, with stabilization occurring after the 14th until the 21st day of treatment.

6 beta-hydroxycortisol in serum and urine as determined by enzyme immunoassay on microtitre plates.

We modified our previous enzyme immunoassay (Clin Chim Acta 1986;157:267-76) for estimating 6 beta-hydroxycortisol (6 beta-OHF) to provide a more routine procedure for measuring it in urine and to increase sensitivity for its measurement in serum. Precision, analytical recovery, specificity, and detection limit are reported. 6 beta-OHF and 17-hydroxycorticosteroids in 24-h and 4-h (08:00-12:00 h) urine collections from healthy volunteers showed no significant day-to-day differences in excretion rate for either collection period. Mean values for 6 beta-OHF in serum of men, women, and women taking oral contraceptives showed no significant differences among these groups.

ELISA of 6-beta-hydroxycortisol in human urine: diurnal variations and effects of antiepileptic therapy.

An enzyme-linked immunosorbent assay for urinary 6-beta-hydroxycortisol (6-beta-OHF) was developed. A highly specific antiserum against 6-beta-OHF-21-hemisuccinate bovine serum albumin (6-beta-OHF-21-HS-BSA) raised in rabbit was used. The enzyme-labelled hapten was prepared by the mixed anhydride method using horse radish peroxidase (HRP). The limit of detection is very low (10 pg), the intra-assay variation ranges from 5.2-6.6%, the inter-assay variation is 8.2-8.3%. Cross reactivities of various steroids are very low. The diurnal variations in urinary 6-beta-OHF and it's ratio to 17-hydroxycorticosteroids (17-OHCS) were studied in six healthy volunteers. No significant difference was found within the 24 h for the ratio of 6-beta-OHF to 17-OHCS. A large increase of 8-12 h urinary 6-beta-OHF was observed in patients receiving chronic treatment with antiepileptics.

Variations in sialic acid content of gamma-glutamyltransferase: a consequence for immunochemical determinations?

Using specific antibodies against the human kidney enzyme, gamma-glutamyltransferase (GGT, EC 2.3.2.2) was assayed from human kidney and serum by electroimmunodiffusion. Determination of the enzyme by such a method was highly influenced by the sialic acid content of the molecule. The peaks corresponding to the sialylated GGT were higher than those corresponding to the neuraminidase-treated enzyme. In contrast, sialylation of the protein had no influence on the results observed when measuring the enzyme by radial immunodiffusion. Moreover, immunoprecipitation curves of both sialylated and neuraminidase-treated samples were identical. The varying degrees of sialylation of GGT occurring under physiological or pathological conditions are known to be partly responsible for the heterogeneity of the enzyme in organs and biological fluids. Therefore, determination of the enzyme by electroimmunodiffusion may be hazardous.

Simultaneous determination of uric acid and creatinine in plasma by reversed-phase liquid chromatography.

Because numerous substances, endogenous compounds as well as xenobiotics, interfere with determination of uric acid and creatinine, we have devised a more nearly specific method by which we can simultaneously determine uric acid and creatinine in plasma by "high-performance" liquid chromatography. We used a mobile phase of ammonium acetate (30 mmol/L) and methanol (156 mmol/L) at pH 7.0 and a flow rate of 1 mL/min. We used a C18 reversed-phase column, and measured absorbance at 235 nm, the wavelength corresponding to the absorption maximum of uric acid and creatinine. Uric acid and creatinine could be determined in less than 5 min by directly injecting plasma diluted fivefold; use of a precolumn eliminated the need for deproteinization. We evaluated the precision, recovery, linearity, specificity, and limit of detection of the method and we checked for analytical interference by 37 currently used drugs. We compared results with those obtained by routine and reference methods.

Associations between serum gamma-glutamyltransferase and apolipoproteins: relationships with hepatobiliary diseases.

We studied the association between gamma-glutamyltransferase (GGT) and apolipoproteins A or B in serum of 42 patients with various hepatobiliary diseases. Binding of the enzyme to apolipoprotein A is not related to a clearly defined disease, but appears to be mainly influenced by the ratio of total cholesterol to GGT activity. An important fraction of GGT activity is associated with apolipoprotein B in patients with icteric or anicteric cholestasis. Conversely, in noncholestatic patients, the percentage of apolipoprotein B-bound GGT activity is low. Addition of the "heavy" form of GGT, obtained by solubilizing the membrane-bound enzyme with detergents, to a serum with low GGT activity led to the binding of the enzyme only to apolipoprotein A. The "light" form of GGT, obtained by limited proteolysis of the "heavy" form and added to the same serum, did not bind to either apolipoprotein A or apolipoprotein B. Thus, the association between the serum enzyme and apolipoprotein A apparently results from nonspecific aggregation of the amphiphilic "heavy" form of the enzyme. The origin of the apolipoprotein B-GGT complexes found in cholestatic patients needs further investigation.

Complexes of serum gamma-glutamyltransferase with apolipoproteins and immunoglobulin A.

We have detected complexes between gamma-glutamyltransferase and apolipoproteins or immunoglobulin A in sera from patients with hepatobiliary diseases but not in sera from healthy individuals. An average of 52.4% of the enzymic activity was precipitated by antiserum against apolipoprotein A, 29.9% by antiserum against apolipoprotein B, and 9.7% by antiserum against immunoglobulin A. Fifty to 60% of the enzyme activity was inhibited in the immunoprecipitates from the transferase fraction bound to apolipoprotein A or immunoglobulin A, and 21% in the fraction bound to apolipoprotein B. We identified the complexed transferase fractions by electrophoresis.

[Multiple molecular forms of serum gamma-glutamyltransferase: characterization and clinical significance]. / Formes moléculaires multiples de la gamma-glutamyltransférase plasmatique: caractérisation et signification clinique.

Heterogeneity of serum gamma-glutamyltransferase was described for the first time about thirty years ago. It seems to result from post-translational modifications of a single type of molecule. The study of that heterogeneity is based upon the use of separation techniques such as gel filtration, ion-exchange chromatography, affinity chromatography with lectines, and especially electrophoresis on various supports. The most promising clinical applications concern hepatology. In particular, appearance of certain fractions observed by electrophoresis on polyacrylamide gel could evoke the existence of malignant phenomena.

A genetic study of serum levels of cobalt-activated acylase among susceptible, resistant and semiresistant strains of mice with experimental viral hepatitis.

Cobalt-activated acylase (Co-A) and transaminase activity were determined in the serum of A/Jax, DBA/2 and C3H mice several days after an intraperitoneal injection of 1,000 lethal doses of murine hepatitis virus type 3 (MHV3). A significant rise in the enzyme activity was observed 1 day after the injection, followed by a decrease on day 2. In the case of the genetically resistant A/Jax strain, the Co-A level regularly decreased to reach normal values on days 7-8. On the contrary, among the fully susceptible DBA/2 strain mice (all dead on day 5), a second rise in acylase (Co-A) level was observed on days 3 and 4, much higher than the day-1 values. Among the mice of C3H strain, which is recorded as 'semi-susceptible', some individuals behaved like the susceptible DBA/2. The comparison of serum acylase activity with other liver function tests showed a correlation between Co-A and transaminases (ALT and AST) with C3H and DBA/2 strains, but no correlation with A/JAX resistant strain. gamma-Glutamyltransferase was not detectable in the serum of different strains during the time of experimentations. Our results suggest that Co-A activity correlates with the clinical course, and that Co-A is a sensitive indicator enzyme in the early phase of viral hepatitis.

The use of gamma-glutamyl-p-aminobenzoic acid as the substrate for determination of gamma-glutamyltranspeptidase activity in blood serum.

By the phthaloyl method less toxic and readily soluble gamma-L-glutamyl-p-aminobenzoic acid was synthesized. This substance was used as a substrate for gamma-glutamyltranspeptidase activity assay in blood serum and urine. Close correlation was shown between the results obtained with the new method and with the old one which used gamma-L-glutamyl-p-nitroanilide.

Acyl derivatives of p-aminobenzoic acid as new substrates for the assay of serum acylase activity.

A simple colorimetric method for the assay of cobalt-activated acylase activity in human serum using new and less toxic N-chloroacetyl-gamma-L-glutamyl-p-aminobenzoic acid as substrate has been described. The values obtained with this method are almost the same as with the previously described method using naphthylamide substrate.

Specific gamma-glutamyl transpeptidase inhibitor from human and animal intestines.

An inhibitor of gamma-glutamyl transpeptidase in human and animal intestines was assayed by means of a new method. In mice fed with LSM fodder, accumulation of the inhibitor in the mucous membrane of the small intestine was observed. Purified gamma-glutamyl transpeptidase inhibitor from mouse and human intestines was identified as L-serine. Neither this amino acid nor purifed inhibitor acted on other intestinal peptidases. Presumably, one of the ingredients of LSM feed influences accumulation of the inhibitor, and consequently diminishes absorption of gamma-glutamyl substrates in the intestine.