Differential action of thyroid hormones and chemically related compounds on the activity of UDP-glucuronosyltransferases and cytochrome P-450 isozymes in rat liver.

The effect of thyroid hormones and chemically related compounds, on the activity of UDP-glucuronosyltransferases (EC 2.4.1.17) and cytochrome P-450-dependent monooxygenases in rat liver microsomes was investigated. The animals were thyroidectomized and treated with different doses of the drugs for 3 weeks. Opposite effects were observed depending on the isoenzyme of UDP-glucuronosyltransferase considered. While 3,3',5-triiodo-L-thyronine, 3,3',5-triiodothyroacetic acid, 3,3',5-triiodothyropropionic acid, isopropyldiiodothyronine and L- and D-thyroxine strongly increased 4-nitrophenol glucuronidation in a dose-dependent fashion, they decreased markedly bilirubin glucuronidation. However, the activity toward nopol, a monoterpenoid alcohol, was not significantly changed regardless of which compound or dose was used. Variation of UDP-glucuronosyltransferase observed with 4-nitrophenol and bilirubin was related to the thyromimetic effect of the drugs estimated from the increase in alpha-glycerophosphate dehydrogenase. Thyronine and 3,5-diiodo-L-tyrosine, which did not enhance this activity, also failed to affect glucuronidation. Variations in UDP-glucuronosyltransferase activity were more likely due to changes in protein expression rather than changes in enzyme latency, since lipid organization of the microsomal membrane, as estimated from the mean anisotropy of 1,6-diphenyl-1,3,5-hexatriene by fluorescence polarization was not significantly modified by the drug administration. Although some of the drugs could significantly decrease the triacylglycerol and cholesterol contents in plasma, all failed to affect lauric acid hydroxylation. The activities of catalase, palmitoyl-CoA dehydrogenase (CN- insensitive) and carnitine acetyltransferase in the fraction enriched in peroxisomes were also not significantly affected by treatment with the thyroid hormone LT3. In contrast, the activity of 7-ethoxycoumarine O-deethylase was increased by large doses of thyronine and by 3,3',5-triiodothyropropionic acid. The concentration of total cytochrome P-450 was decreased in a dose-dependent fashion by all the compounds used, except thyronine. Finally, significant correlations were observed between glucuronidation of bilirubin and 4-nitrophenol and the content in cytochrome P-450. This suggests a possible coordinate regulation of the two processes, which depends on the physicochemical characteristics of the thyroid hormones and related compounds.

Opposite regulation of bilirubin and 4-nitrophenol UDP-glucuronosyltransferase mRNA levels by 3,3',5 triiodo-L-thyronine in rat liver.

The effects of 3,3',5 triiodo-L-thyronine (L-T3) on the constitutive levels of hepatic mRNA encoding two UDP-glucuronosyltransferase (UGT) isoforms implicated in the glucuronidation of planar phenolic substrates (UGT1*06) and bilirubin (UGT1*0) were investigated in rat liver. The amount of UGT mRNA was quantitated by reverse transcription and amplification methods (RT-PCR). Treatment with L-T3 significantly increased UGT1*06 and decreased UGT1*0 mRNA levels by 41% and 54%, respectively. The opposite situation was observed in thyroidectomised animals. A good relationship observed between UGT activity toward 4-nitrophenol and bilirubin and mRNA levels emphasizes the key role played by the thyroid hormone L-T3 on UGT expression.

Comparative quantification of two hepatic UDP-glucuronosyltransferase bilirubin isoforms mRNAs in various thyroid states in rat.

The study was designed to compare the effects of 3,5,3' triiodo-L-thyronine (L-T3) on the levels of hepatic mRNAs encoding two UDP-glucuronosyltransferase bilirubin isoforms (UGT1*1 and UGT1*0) in rats, by reverse transcription and quantitative polymerase chain reaction (RT-PCR). The administration of L-T3 decreased the UGT1*O mRNA by 2.2-fold and that of UGT1*1 by only 1.4-fold. In contrast, thyroidectomy increased the UGT1*O mRNA level by twofold but did not change that of the UGT1*1 isoform significantly. Interestingly, treatment with a known inducer of UGT bilirubin, ciprofibrate, induced the hepatic mRNA levels encoding for the UGT1*0 isoform by 3.5-fold and for the UGT1*1 isoform by only twofold. The results indicate for the first time that, although UGT1*1 mRNA is indeed a major transcript, its level is weakly affected by these compounds. In contrast, the minor UGT1*0 form is much more sensitive both to the action of this drug and to changes in thyroid status. The data support the notion that the various members of exon1 of the UGT1 locus have their own individual regulatory region.

Liver microsome phospholipids and cytochrome P.450 concentration in phenobarbital treated rats fed on different diets.

Liver microsomal concentration of cytochrome P.450 is increased in animals which are fed diets rich in polyunsaturated fatty acids. On the other hand, the effects of phenobarbital are more important when the dietary fat is more unsaturated. The unsaturation index in liver microsomal phosphatidylcholines depends on the unsaturation of the dietary fats. The treatment with phenobarbital constantly results in a decrease of the unsaturation index of fatty acids both in lecithins and cephalins. The importance of the liver microsomal cytochrome P.450 increase and the importance of the unsaturation index decrease in liver microsomal lecithins, both promoted by phenobarbital, are in good agreement.

Inductive effects of fenofibrate and metabolism of phenobarbital.

The inductive effects of fenofibrate (FF) and phenobarbital (PB) were investigated in male Wistar rats. FF treatment produced an inductive effect on liver weight, cytochrome P450 content, and aniline hydroxylase (AH) and bilirubin UDP-glucuronosyltransferase (UDP-GT) activities in liver microsome fraction. PB and FF inductive effects were additive on liver weight but were not additive on P450 microsomal concentrations. On the contrary, FF administration decreased the inductive effect of PB on bilirubin UDP-GT activity. When FF and PB treatment were coupled, plasma and liver PB concentrations were not affected, whereas OHPB concentrations, especially in liver homogenate, were greatly decreased. Thus it can be concluded that the production of OHPB from PB was probably not accelerated, but the elimination of OHPB, the main metabolite of PB, was considerably enhanced. These results are to be compared with recent reports of structure-dependent induction of bilirubin glucuronidation by arylcarboxylic acids chemically related to clofibrate.

6-Mercaptopurine pharmacokinetics after use of azathioprine in renal transplant recipients with intermediate or high thiopurine methyl transferase activity phenotype.

Prevention of allograft transplant rejection by the immunosuppressive 6-thiopurine drug azathioprine is limited by haematological toxicity (leucopenia or agranulocytosis). This toxicity is particularly apparent in subjects with low thiopurine methyltransferase activity (TPMTase) phenotype (1% in the Caucasian population). The thiopurine derivative 6-mercaptopurine is the active metabolite of azathioprine, and it would be of interest to measure, after validation of plasma measurements, the mean values of the pharmacokinetic parameters in transplant patients with high or intermediate TPMTase phenotypes (85 and 14% of the Caucasian population, respectively). We measured erythrocyte TPMTase activity in 103 kidney transplant recipients of high or intermediate phenotype and calculated, after a test dose of azathioprine, the mean values of the pharmacokinetic parameters for 6-mercaptopurine. We also compared these values with the same parameters from one subject with low TPMTase activity phenotype. The mean observed area under the plasma concentration-time curve (AUC) was 190+/-140 ng mL(-1) h and the elimination rate constant (Kel) was 1.92+/-1. The pharmacokinetic parameters (AUC, Kel, t1/2el (the elimination half-life)) of 6-mercaptopurine in transplant patients are normally distributed and suitable for acceptance as a gold standard value for this population of Caucasian transplant patients. It seems useful to calculate these parameters, representative of the systemic exposure of individual patients to the drug, before prescribing these subjects azathioprine immunosuppressive treatment. In subjects with low TPMTase phenotype these pharmacokinetic measurements could also be an index of dose reduction.

Nutritional factors of microsomal enzymatic induction: influence of lipidic components of the diet.

We compared the ability of two different diets containing 6 per cent of maize oil and 6 per cent of fish oil to modify: firstly the enzyme induction by phenobarbital and secondly the phenobarbital hydroxylation by the liver either in vivo or during in vitro perfusions. The presence of fish oil in the diet increased the cyt P 450 content and the bilirubin glucuronosyl transferase activity. The two induction effects promoted by the association of the phenobarbital treatment and the eating of the fish oil were not additive and it was found that the phenobarbital induction effect was decreased by the fish oil consumption. Phenobarbital and p-hydroxyphenobarbital kinetics were different in the two groups of animals. Phenobarbital was more slowly eluted in the fish oil fed than in the maize oil fed rats while p-hydroxyphenobarbital was more slowly eluted by the fish oil-fed rat livers.

Cyclosporine determinations in heart and liver transplant recipients: comparison of HPLC and FPIA methods on whole blood and plasma.

One hundred and eighty five whole blood samples were analysed for cyclosporine levels by fluorescence polarization immunoassay (FPIA) and high performance liquid chromatography (HPLC). 123 came from 4 heart transplant recipients (mean age +/- SD: 47.50 +/- 20.56 years) and 62 from 4 liver transplant recipients (44.50 +/- 16.52 years). FPIA was done on plasma and whole blood in heart transplant recipients, on plasma in the liver recipients. HPLC was always done on whole blood. The results show a good correlation between FPIA on plasma (y) and HPLC (x) in liver recipients (n = 62, r = 0.935, y = 1.23x + 70 ng/ml), slightly worse between FPIA on plasma (y) and HPLC (x) in heart recipients (n = 64, r = 0.610, y = 0.78x + 189 ng/ml) and mediocre for FPIA on whole blood (y) and HPLC (x) in heart recipients (n = 123, r = 0.566, y = 1.35x + 594 ng/ml).

[Local anesthetics: simultaneous HPLC determination of lidocaine (Xylocaine) and bupivacaine (Marcaine) in human serum]. / Anesthésiques locaux: dosage simultané par CLHP de lidocaïne (Xylocaïne) et de bupivacaïne (Marcaïne) dans le sérum humain.

In order to monitor loco-regional anaesthesia in the upper limbs with a combination of two anaesthetics, the authors participated in a clinical pharmacology study. They describe a method of simultaneous assay of bupivacaine and lignocaine by direct phase HPLC. In particular, the use of heptane sulfonic acid (counter-ion) in the mobile phase allows the elution of the local anaesthetics to be controlled. The criteria of reliability studied enable this technique to be considered for routine use.

Methods of theophylline assay and therapeutic monitoring of this drug.

The purpose of this article is to review various analytical methods of monitoring plasma theophylline. This article was investigated by the "Drug Commission" of SFBC (Société Française de Biologie Clinique). The primary objective is to provide the "know-how", particular for this analysis, which allows the choice between various analytical methods available: immunochemical or physiochemical ones. The techniques described are not necessarily the best, they are approved and tested methods which are the most frequently used in routine practice. The proposed immunochemical methods are: absorption spectroscopy methods: Enzyme ImmunoAssay (EIA), Enzyme Multiplied ImmunoAssay Technique (EMIT); Reflectance spectroscopy method: Apoenzyme Reactivation Immunoassay System (ARIS); Fluorometry spectroscopy method: Substrate Labeled FluoroImmunoAssay (SLFIA); Fluorometry spectroscopy on solid base; Polarization fluorescence spectroscopy ImmunoAssay (FPIA); Turbidimetric measurements: Particle Enhanced Turbidimetric Inhibition ImmunoAssay (PETINIA); Nephelometric measurement: Nephelometric Inhibition ImmunoAssay (NIIA). And the proposed physicochemical methods are: High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC). The second objective is a review of pharmacological properties of theophylline, necessary for a good understanding of therapeutic drug monitoring: intestinal resorption, distribution, metabolism and elimination, drug interactions, dose/response relationship, physiopathological variations and proposed "predictive" "theophylline test". The authors conclude that because of the multiplicity of methodologies used in theophylline therapeutic monitoring the choice of one of them is not easy. The best way to compare different techniques available would be the use of a "reference material" for theophylline monitoring and a quality control network between different clinical pharmacological laboratories.

[Determination of serum or plasma alpha-tocopherol by high performance liquid chromatography: optimization of operative models]. / Dosage de l'alpha-tocophérol sérique ou plasmatique par chromatographie liquide haute performance: optimisation du mode opératoire.

A previous multicentric study set up by the Société française de biologie clinique has emphasized the usefulness of a standardized procedure for the determination by high performance liquid chromatography of alpha-tocopherol in serum or plasma. In our study, we have tested every step of the different published procedures: internal standard adduct, lipoprotein denaturation and vitamin extraction. Reproducibility of results was improved by the use of tocol as an internal standard when compared to retinol or alpha-tocopherol acetates. Lipoprotein denaturation was more efficient with ethanol addition than with methanol and when the ethanol/water ratio was > or = 0.7. Use of n-hexane or n-heptane gave the same recovery of alpha-tocopherol. When organic solvent/water ratio was > or = 1, n-hexane enabled to efficiently extract, in a one-step procedure, the alpha-tocopherol from both normo and hyperlipidemic sera. Performances of the selected procedure were: detection limit: 0.5 microM--linear range: 750 microM--within run coefficient of variation: 2.03%--day to day: 4.76%. Finally, this pluricentric study allows us to propose an optimised procedure for the determination of alpha-tocopherol in serum or plasma.

[Effects of several antilipemic agents on the activity of liver microsomal enzymes]. / Effets de divers hypolipoprotéinémiants sur l'activité des enzymes microsomales hépatiques.

Male Wistar rats were treated daily for 7 days with clofibrate (250 mg/kg/d), benfluorex (50 mg/kg/d), tiadenol (200 mg/kg/d), nicoclonate (100 mg/kg/d) or hexanicit (50 mg/kg/d). The cytochrome P 450 level and ethoxycoumarin deethylase activity (ECDE) in liver microsomes were markedly increased by administration of clofibrate and slightly increased by tiadenol. Benfluorex only increased the activity of ECDE and nicoclonate and hexanicit had no effect. Clofibrate, tiadenol and benfluorex increased the activity of microsomal bilirubin UDP-glucuronosyltransferase. On the other hand, the nicotinic derivatives were ineffective. Tiadenol clearly enhanced the inductive effects of phenobarbital.

[Influence of 3,5,3'-triiodo-L-thyronine on the hepatic enzyme activities responsible for the metabolism of xenobiotics during the development of the chick embryo]. / Influence de la 3,5,3'-triiodo-L-thyronine sur les activités enzymatiques hépatiques responsables du métabolisme des xénobiotiques pendant le développement de l'embryon de Poulet.

The influence of thyroid hormones on microsomal drug metabolizing enzymes was studied in hypothyroid newborn rats and chick embryos. Administration of 3,5,3'-triiodo-L-thyronine strongly decreased the microsomal cytochrome P 450 content in hypothyroid new-born rats and thus could render the rat pup more susceptible to hepatotoxicity from drugs. The drug metabolizing system in 20 days old chick embryos was less sensitive to the effects of thyroid hormone, but administration of phenobarbital was accompanied by a strongly induction effect on microsomal enzyme activities.

[Inductive effects of enzymes of salicylic acid derivatives]. / Effets inducteurs d'enzymes des dérivés salicylés.

In the series of salicylic acids derivatives investigated 3,5-DIS has a large inductive effect of enzymes with respect to the metabolism of xenobiotics: rise in the rate of Cyt. P 450; increase of ethoxycoumarin deethylase, bilirubin glucuronosyl transferase and benzphetamine-N-demethylase activities. This effect is comparable with that of phenobarbital.