[Determination of reference values: a priori or a posteriori selection?].
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Adult↗
Biomedical subjects
G Siest
Publications and source records attributed to G Siest.
Structure-dependent induction of bilirubin glucuronidation and lauric acid 12-hydroxylation by arylcarboxylic acids chemically related to clofibrate.
The inductive potency of carboxylic acids, structurally related to clofibrate, on bilirubin UDPglucuronosyltransferase was investigated in the rat. For this purpose, structure-induction relationships were established using ten different arylcarboxylic or chlorophenoxycarboxylic acids. 4'-Chlorophenoxyacetic, -propionic and -isobutyric (clofibric) acids progressively increased hepatic glucuronidation of bilirubin (17%, 43%, 60% greater than controls, respectively) after a 5-day treatment in rat (100 mg/kg per day). 2-Phenylpropionic acid also enhanced bilirubin UDPglucuronosyltransferase activity (50%) in contrast to phenylacetic acid. The other compounds did not, or only slightly, affect this parameter. These results indicate that specific structural features are required for the induction property. Moreover, a good correlation (r = 0.962) was found between the extent of induction and the physiochemical descriptors which characterize the electronic state of the molecules, when analysed by multidimensional regression. Fluorescence polarization revealed that the compounds tested, especially clofibric acid, did not affect, in vivo or in vitro, the anisotropy of two different probes embedded in the microsomal membranes. Finally, since the interaction of the carboxylic acids with the membranes did not modify the latency state of bilirubin UDPglucuronosyltransferase, it was concluded that the increase in enzyme activity was due more to a real induction than to activation of bilirubin UDPglucuronosyltransferase. A close linkage was established between bilirubin UDPglucuronosyltransferase induction and that of cytochrome P-452, as shown by enhanced omega-oxidation of lauric acid. This led to the hypothesis that both processes could be under coordinate regulation and mediated by a molecular interaction depending on the physicochemical properties of the carboxylic acids.
Animals↗
Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase activities. Conjugations of phenolic and monoterpenoid aglycones in control and induced rats and guinea pigs.
In this report we present evidence that the heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) (UDPGT) activities depends on the chemical structures of the aglycones as well as their biophysical constants. Three animal models were used: Wistar rats, which have active UDPGTs; Gunn rats, in which some of the UDPGT activities are reduced, but which can be induced by phenobarbital; and guinea pigs. In Wistar rats, we found that some coumarins were poor substrates of UDPGT (GT1) and that twenty monoterpenoid alcohol activities showed typical phenobarbital-inducible behavior. In Gunn rats, we showed that substitution of the phenolic aglycone by bulky (alkyl- or methoxy-) groups in the 2-position of the phenolic ring decreased UDPGT (GT1) activity, whereas substitution in the 4-position resulted in an increase in this activity. We also showed that, in this particular strain, activities toward terpenes were less affected than activities toward flat (aromatic) aglycones. Induction by phenobarbital in Gunn rats increased the activity and limited the deficiency for monoterpenoid alcohols. In guinea pigs, we confirmed that phenobarbital selectively increased the activities of UDPGT towards twenty monoterpenoid alcohols without affecting other typical phenobarbital-induced activities such as those for conjugation of morphine. Finally, we showed that orientation of the aglycone molecule in the active site was apparently related to its dipole moment and that the distance between "acceptor-oxygen" (hydroxyl) and the carbons out of the general plane of the molecule was an important factor. These studies clearly suggest that rat and guinea pig contain a UDPGT(monoterpenoid alcohols) with restricted specificities and also that UDPGT(GT1) comprises at least two or three different isoenzymes, each with a slightly different restricted specificity towards flat aromatic aglycones.
Animals↗
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.
Acyltransferases↗
Plasma uric acid: mass spectrometric method for control and comparison of intra- and interlaboratory studies.
We propose a preliminary study for the utilization of isotope dilution and mass fragmentography for the measurement of plasma uric acid. Using stable isotopes it is possible to measure this compound in comparison with the molecule labelled with three 15N atoms (1,3,9). We achieved the determination at the molecular peak. A linear calibration curve is obtained from standard solutions between 0 and 600 mumol/l. The method has good precision and the results obtained with different quality control materials demonstrate the need for better accuracy. This method could, after more detailed studies, be a good candidate for a definitive method.
Gas Chromatography-Mass Spectrometry↗
Conformational studies of NADPH cytochrome P-450 reductase by circular dichroism: interaction with phospholipids.
Ultraviolet circular dichroism spectrum of purified NADPH cytochrome P-450 reductase was characterized by two negative bands centered at 208 and 222 nm. The approximation of the alpha-helical content from the value of the mean residue ellipticity at 222 nm indicated 28% of alpha-helical structures. Heat inactivation of the enzyme was associated to a drastic change in the secondary structure of the protein. Membrane reconstitution experiments by inclusion of the enzyme into liposomes revealed that the conformation of NADPH cytochrome P-450 reductase was sensitive to its phospholipid environment. Egg lecithin as well as synthetic phosphatidylcholines, at the optimal phospholipid-enzyme molar ratio 200, was able to increase up to 37% the mean residue ellipticity at 222 nm. Addition of phosphatidylserine or phosphatidylethanolamine produced no effect. Non-ionic detergent such as Emulgen 913 weakly enhanced the mean residue ellipticity.
Animals↗
UDP-glucuronosyltransferase(s) activities towards natural substrates in rat liver microsomes. Kinetic properties and influence of triton X-100 activation.
We studied the in vitro capability of hepatic microsomal UDP-glucuronosyltransferase (UDPGT) in male rats to conjugate 22 natural xenobiotics which are known to be excreted as glucuronides in vivo. We clearly demonstrated that the Vmax can range in a decreasing scale for the following families of aglycones: 7-hydroxylated coumarins greater than 2-naphthol and phenols greater than monoterpenoid alcohols greater than 4-hydroxylated coumarins. The Km app. cannot be arranged in the same scale. This suggests that the catalytic mechanism of UDPGT is dependent on the hydroxyl group reactivity rather than on the binding interaction at the active site expressed by the Km app. The effects of various concentrations of detergent (Triton X-100) were determined on specificity (apparent Km) and activity (Vmax). For the 22 aglycones we showed that activation caused a variation in the Vmax which was a function of the concentration in detergent. The maximum of this activation did not always correspond to the same detergent/protein weight ratio. The impact of activation on Km app. was less clear since the variations observed were slightly different.
Animals↗
[Effect of thyroid status on microsomal enzymes during liver regeneration in the rat].
The effects of thyroid status upon cyt. P 450 concentration and ethoxycoumarin deethylase, benzopyrene hydroxylase and UDP-glucuronosyltransferase activities in the liver microsome fraction were far more important in partially hepatectomized rats than in control animals. The partial hepatectomy simultaneously lowered the MFO enzymes activities in the hepatic microsome fraction and made them more sensitive to thyroid hormones effects.
7-Alkoxycoumarin O-Dealkylase↗
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.
Autoanalysis↗
Total cholinesterase in plasma: biological variations and reference limits.
Factors affecting biological variation in total cholinesterase activity in plasma were studied in a population of 3372 apparently healthy subjects at least four years old. Determination of the dibucaine number, after inhibition of the activity by dibucaine, made it possible to specify the contributions of genetic and physiological characteristics to variations in the total activities by using a statistical method of segmentation. The main factors modifying plasma cholinesterase activity in males are genetic status (dibucaine number) and degree of overweight (subscapular skinfold). In females, hormonal status (puberty or menopause), genetic status (dibucaine number), and the use of oral contraceptives are the factors mainly responsible for the variations of plasma cholinesterase activity. These data allow us to propose reference limits for total plasma cholinesterase.
Adolescent↗
Variations in total phospholipids and high-density lipoprotein phospholipids in plasma from a general population: reference intervals and influence of xenobiotics.
The influence of different factors on variations in the concentrations of total phospholipids and high-density-lipoprotein (HDL) phospholipids in plasma was studied in a presumably healthy population of 2000 subjects, four to 70 years old. Age is the major factor associated with variation of total phospholipids. In females, this is due in part to age-related changes in hormonal status. The use of oral contraceptives affects only HDL phospholipid values. Use of tobacco does not influence plasma phospholipid values, but alcohol consumption increases values for both total and HDL phospholipids. We propose reference intervals for total plasma phospholipids and HDL phospholipids, adjusted for age and sex. Screening for lipid status can now include determinations of phospholipids as well as cholesterol and triglycerides.
Adolescent↗
Differential solubilization of rabbit liver plasma membrane gamma-glutamyltransferase by proteases and detergents: effect of phenobarbital treatment.
In an attempt to understand the mechanism of gamma-glutamyltransferase transfer from the liver to the plasma, potassium chloride, sodium dodecyl sulfate and proteases (papain and bromelain) were used to solubilize rabbit liver plasma membrane gamma-glutamyltransferase. Potassium chloride solutions solubilized 10-30% of membrane proteins but only 1-3% of membrane gamma-glutamyltransferase activity. However, when sodium dodecyl sulfate is used, even at low concentration (0.1-0.2%, w/v) greater than 90% of membrane gamma-glutamyltransferase activity and about 80% of membrane proteins can be solubilized. Furthermore, we showed that unlike the effect of bile salts on the membrane gamma-glutamyltransferase of phenobarbital-treated animals, the same treatment seems to have no influence on membrane gamma-glutamyltransferase solubilization by proteases. Indeed, the ratios of gamma-glutamyltransferase solubilization by papain or bromelain were the same for liver membranes obtained from control and phenobarbital-treated animals.
Animals↗
Structural changes of liver microsomes in rat during neonatal life: influence on the glucuronidation rates of various substrates.
The Vmax of the membrane bound UDP-glucuronosyltransferase (UDP-GT) towards group 1 substrates (4-nitrophenol, 2-naphthol) was particularly higher in young rats than in adults. On the contrary, activity towards group 2 substrates such as borneol or testosterone was not detectable in fetus liver. The developmental pattern of UDP-GT was related to changes in lipid composition of microsomes, namely in the content in lysophosphatidylcholine which rose at birth. The phospholipid-cholesterol molar ratio also increased 2 fold from the 16th day of fetal life to the 4th day after birth. Measurement of the steady state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as determination of the order parameter S and the rotation cone angle of the fluorescent probe strongly suggested an increase in membrane fluidity in rat liver microsomes during ontogenesis.
Animals↗
Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) activities: comparison between human and mammalian species activities.
The first comparative profiles of UDP-glucuronosyltransferase(s) (UDPGT) activities obtained under standard conditions in vitro in mammals (man, rat [Wistar and Gunn], mouse, monkey [Papio papio and Cynomolgus], pig, guinea pig, rabbit, dog) are presented for 16 aglycones. A decreasing scale of these activities was obtained from planar to bulky molecules. The scale was identical for each of the mammals studied, including man. Statistical analysis of the results revealed a division of the aglycones into three groups, one being correlated with the molecular form called GT1 the two others with the GT2 form. The profile of activities in the Gunn rat revealed very weak activity towards planar molecules (GT1). These results provide evidence that under standard conditions, human UDPGT activities are comparable to those from other animals.
Animals↗
Assessment of the Mulder and Van Doorn kinetic procedure and rapid centrifugal analysis of UDP-glucuronosyltransferase activities.
The optimal experimental conditions of the enzyme assay described by Mulder and Van Doorn (1975, Biochem J. 151, 131-140) for the measurement of UDP-glucuronosyltransferase activities were tested towards structurally different aglycones. This assessment of this assay revealed that addition of Triton X-100 as enzyme activator was necessary because of its apparent inhibitory effects on interfering reactions. Under these conditions, accordance of the data with results published in the literature was obtained. We present for the first time an UDP-glucuronosyltransferase assay adapted on a fast analyser centrifuge which allows a rapid and sensitive measurement of enzyme activity that is very useful for kinetic constant determination, without consuming a large volume of reagents.
Animals↗
Inhibition studies of microsomal UDP-glucuronosyltransferase activities by furosemide and salicylamide.
Contrarily to cytochrome P-450, a few inhibitors of UDP-glucuronosyltransferase have been described. We verified the nature of the in vitro inhibition due to furosemide, using 4 different aglycones (morphine, p-nitrophenol, borneol and eugenol) presumably belong may to different clusters of UDP-glucuronosyltransferase activities. The variations of these corresponding kinetic parameters (Km, Vmax, specific activities) must correspond to different inhibition mechanisms of furosemide, for example different site(s) of fixation in the area of the active site of UDPGT. Because these variations were not as classically described, we checked the impact of furosemide pretreatment on in vitro levels of different UDPGT activities. We compared these result, with another inhibitor (salicylamide). The apparent induction due to the both molecules enforced the hypothesis of a complexe inhibition mechanism.
Animals↗
Kinetic properties of rat liver UDPglucuronosyltransferase during ontogenesis.
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Age Factors↗
Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver.
Two microsomal epoxide hydrolases of the rat liver were found to be differentially regulated in hyperplastic nodules. Whilst the activity for substrates of the well-known microsomal epoxide hydrolase with a broad substrate specificity (EHb), benzo[a]pyrene 4,5-oxide and androstene oxide (16 alpha, 17 alpha-epoxyandrosten-3-one), was greatly (approximately 5-fold) increased in the nodule microsomes and moderately (approximately 2-fold) increased in the surrounding tissue, that for the substrate of the novel microsomal epoxide hydrolase, cholesterol 5 alpha,6 alpha-oxide (EHch) remained unchanged. Since both enzymes convert endogenous steroid epoxides but with distinct structural features, this differential regulation may indicate a role of endogenous steroid epoxide(s) of a defined structure during hepatocarcinogenesis. Alternatively, this differential regulation may serve as a marker during hepatocarcinogenesis.
Animals↗