Weak relationships between blood antioxidant enzymes and serum lipids or apolipoproteins.
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Adolescent↗
Biomedical subjects
G Siest
Publications and source records attributed to G Siest.
In vivo study of the elimination from rat brain of an intracerebrally formed xenobiotic metabolite, 1-naphthyl-beta-D-glucuronide.
Among the drug-metabolizing enzymes present in the rat brain, one form of UDP-glucuronyltransferase catalyzes the formation of the polar metabolite 1-naphthyl-beta-D-glucuronide from 1-naphthol. We measured the activity of this isoform in different brain regions and showed its heterogeneous distribution. Conjugation activities were found to be the highest in the olfactory bulbs (25.4 nmol/h/mg protein) and lowest in the cerebellum (4.5 nmol/h/mg protein). As the blood-brain barrier prevents the passage of hydrosoluble molecules, we studied in vivo the characteristics of the efflux of labeled 1-naphthyl-beta-D-glucuronide injected into the lateral ventricle and the cortex tissue, using tritiated water and labeled inulin as reference compounds. The results reported here indicate that intracerebrally formed glucuronide is cleared from brain tissue by both diffusion and a saturable efflux process.
Animals↗
Highly sensitive immuno-assays for the determination of cotinine in serum and saliva. Comparison between RIA and an avidin-biotin ELISA.
Two immuno-assay methods (RIA and ELISA) have been developed for the accurate and sensitive measurement of cotinine in human body fluids (serum, saliva). RIA uses [3H]cotinine as antigen and charcoal/dextran for separating cotinine-bound antibodies from the free derivative. Another technique (ELISA) was developed to avoid the use of radio-labelled compounds and to determine cotinine in large populations, including passive or non-smokers who usually present very low concentrations. The two techniques were analytically validated. The detection limit was similar (0.1 micrograms/l) and the precision was better than 10% for both techniques. Non-smoker values ranged from 0.1 to 17 micrograms/l by ELISA and 0.1 to 27.5 micrograms/l by RIA, whereas smoker values ranged from 50 to 1000 micrograms/l (ELISA) and from 70 to 800 micrograms/l (RIA). The comparative analysis of cotinine in 96 human sera revealed a good correlation between the two methods (r = 0.97) and a reliable discrimination between the populations of non-smokers and smokers. As usual, the ELISA is more rapid (4 h 30 min) than the RIA (longer than 48 h). ELISA is proposed for use in the epidemiological investigation of the human tobacco risk.
Avidin↗
Signal peptide-length variation in human apolipoprotein B gene. Molecular characteristics and association with plasma glucose levels.
We studied the molecular characteristics of three naturally occurring variants in the human apolipoprotein B (apoB) signal peptide, their frequencies in non-insulin-dependent diabetic and random populations, and their association with several measures of lipid and carbohydrate metabolism. In a random sample of 197 French whites, there were two common alleles, 5'beta SP-24 and 5'beta SP-27, with frequencies of 0.35 and 0.65, respectively. In a random sample of 181 Mexican Americans, there was an additional allele, 5'beta SP-29, with a frequency of 0.03. DNA sequence analysis indicated that the signal peptide alleles consisted of the following: 5'beta SP-29 encoded 29 amino acids in the signal peptide containing two copies of the sequence CTG GCG CTG encoding Leu-Ala-Leu and a consecutive run of eight Leu-encoding codons; 5'beta SP-27 encoded 27 amino acids with a run of only six Leu codons; 5'beta SP-24 encoded 24 amino acids and contained a single copy of CTG GCG CTG and a run of six Leu codons. In the sample of French whites, average apoAI and glucose levels were significantly different among signal peptide genotypes. 5'beta SP-24/24 homozygotes had higher apoAI levels than the two other signal peptide genotypes (1.59 vs. 1.42 g/L, respectively). Heterozygous 5'beta SP-24/27 individuals had the highest glucose levels. In the random sample of Mexican Americans, average glucose levels were also significantly different among signal peptide genotypes. However, the rank order of average glucose levels was not the same between the two samples.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Identification of novel phencyclidine metabolites formed in vitro by rabbit microsomal metabolism.
1. Phencyclidine (PCP) was incubated with rabbit liver and brain microsomal fractions, and the structures of metabolites formed by oxidation determined by g.l.c.-mass spectrometry. 2. The formation of several known mono- and di-hydroxylated metabolites, as well as two new metabolites, was seen in the liver preparations. 3. Hydroxylated PCP metabolites were also formed after incubation of PCP with brain microsomes, indicating that PCP biotransformation may occur in the brain itself.
Animals↗
Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae.
To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.
Animals↗
Stable expression of two human UDP-glucuronosyltransferase cDNAs in V79 cell cultures.
Two human liver UDP-glucuronosyltransferase cDNA clones (HLUGP1 and HLUG25) were individually inserted into the eukaryotic expression vector pKCRH2. Each recombinant plasmid was cotransfected with a SFVneo vector, thereby allowing establishment of several V79 cell lines retaining the exogenous UDP-glucuronosyltransferase cDNA after selection with G418 (Geneticin). Southern blot analysis suggested that the cDNAs were integrated into the host cell genome. Northern blot and immunoblot analyses indicated that the cDNAs were correctly transcribed and translated for the production of functional enzymes. The established recombinant V79 cell lines stably expressed the UDP-glucuronosyltransferase activities towards 1-naphthol (HLUGP1) and hyodeoxycholic acid (HLUG25) at levels 10-20-fold higher than with transient expression, and in the range found in human liver. These high levels of expression of UDP-glucuronosyltransferase activity allowed the determination of apparent kinetic constants and substrate specificities of glucuronidation in the genetically engineered cell lines. HLUG25 cDNA encoded an isoform with restricted specificity towards the 6-OH group of the bile acid hyodeoxycholic acid. The other steroids, bile acids, endobiotics, and xenobiotics tested as substrates were glucuronidated in various samples of human liver microsomes, but not by this isoenzyme. This study, allowing the expression of individual UDP-glucuronosyltransferases in heterologous cells with no endogenous transferases, offered a unique solution for the characterization of UDP-glucuronosyltransferase functional heterogeneity.
Animals↗
Biological variability of superoxide dismutase, glutathione peroxidase, and catalase in blood.
We studied the biological variability of blood superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GPX; EC 1.11.1.9), and catalase (CAT; EC 1.11.1.6) in a sample of 1836 apparently health subjects, ages 4-97 years. SOD and GPX activities were assayed in plasma (P) and erythrocytes (E) by automated methods, and CAT was measured in erythrocytes by a manual technique. No statistically significant variation of these antioxidant enzyme activities according to gender was demonstrated, except for E-GPX, which was slightly but significantly higher in women than in men (P less than 0.001). Activities appear rather stable in adults less than 65 years old, but decrease for most enzymes in the elderly. There is no evidence that weight, blood pressure, or menopause influences the antioxidant enzymes' activities. In girls ages 10-14 years, E-SOD activity is reduced by 16% (P less than 0.05) after menarche. Variations related to smoking and alcohol consumption are slight and concern only P-SOD and P-GPX, respectively. Conversely, intake of some drugs (e.g., anti-inflammatory agents, antidepressants, and thyroid hormones) modifies activity of some of the three enzymes. E-SOD positively correlates with P-SOD (r = 0.216, P less than 0.001) and E-CAT (r = 0.123, P less than 0.001), and E-GPX with P-GPX (r = 0.218, P less than 0.001). Finally, we propose reference intervals for activities of the three antioxidant enzymes in blood in individuals less than 65 years old.
Adolescent↗
Relation between debrisoquine oxidation phenotype and morphological, biological, and pathological variables in a large population.
Factors affecting biological variations in debrisoquine-oxidation polymorphism were investigated in a population of 3065 unrelated supposedly healthy Caucasian subjects, ages 35 to 50 years. This population, including 1526 men and 1539 women, was used to determine whether the debrisoquine-oxidation phenotype can be related with environmental factors such as alcohol intake, smoking habits or medication; with morphological variables; or with 22 blood constituents and some pathological states. A single dose of 10 mg of debrisoquine sulfate was administered to determine the oxidation phenotype. A metabolic ratio (debrisoquine/4-hydroxydebrisoquine) of 10.0 defined a poor metabolizer [frequency of 8.2% (SD 1.0%)] in this sample. Dose recoveries of debrisoquine excretion (mean and SD) were 8.9% (11.9%) and 45.1% (32.2%) in extensive and poor metabolizers, respectively. The mean (SD) amount of debrisoquine administered that was excreted in urine as 4-hydroxydebrisoquine was 17.4% (17.3%) in extensive metabolizers and 0.5% (0.9%) in poor metabolizers. The main factors differing significantly between poor and extensive metabolizers were mean cell volume, mean corpuscular hemoglobin concentration, albumin, and ponderal index. No other blood constituents (e.g., cholesterol, glucose) differed between poor and extensive metabolizers. The lack of correlation with most of the variables tested is of interest in clinical trials, because our findings indicate that no subgroups will be required, making selection of subjects easier.
Administration, Oral↗
Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers.
After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase. We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine. These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture. The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized. Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated. Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE. Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum). It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.
Blotting, Western↗
Distribution of cytochrome P450 activities towards alkoxyresorufin derivatives in rat brain regions, subcellular fractions and isolated cerebral microvessels.
The regional and subcellular distributions of rat brain cytochrome P450 and cytochrome P450-dependent activities were examined. Cytochrome P450 was found to be mainly localized in mitochondria in all the six cerebral regions studied. The activities of the isoforms mostly implicated in drug metabolism, cytochromes P450 b and c, were measured by the dealkylation of two alkoxyresorufins, that are sensitive probe substrates for these isoforms. These activities have been measured in microsomal and mitochondrial fractions obtained from six different regions in male rat brains, as well as in microvessels. Resorufin derivatives dealkylation specific activities were higher in brain microsomal fractions than in hepatic ones in all the six regions examined when results were expressed per cytochrome P450 content. These brain microsomal specific activities were also higher than in mitochondrial fractions. Olfactory bulbs showed the highest cytochrome P450 content and activities in both microsomal and mitochondrial fractions. A sex-linked difference in cytochrome P450-dependent activities was also found. After an in vivo inducing pretreatment of rats, only 3-methylcholanthrene induced ethoxyresorufin O-deethylase activity, in the three preparations studied. These results provided (i) direct evidence that cytochromes P450 b and c isoforms are active in brain microsomal fractions, with regional and sex-linked differences, and (ii) the first demonstration of cytochrome P450-dependent activities in isolated rat brain microvessels.
Animals↗
Enzyme immunoassay for flecainide.
In order to develop an enzyme immunoassay (ELISA) for measurement of plasma flecainide levels, we first synthesized flecainide-hemisuccinate-BSA as immunogen. A highly specific antiserum was then raised in rabbits. Enzyme-labelled flecainide was prepared by the mixed anhydride method using hemifumarate-flecainide and horse radish peroxidase. The limit of detection was 10 ng/ml, the method was linear up to 1,000 micrograms/l. The intra and inter-assay variation was 5.1-7.8%. Cross reactivity with various metabolites of flecainide or some chemically related drugs was less than 0.1%. By contrast the cross reaction with some structural analogues achieved 25%. The importance of parts of the aromatic ring structure in immunogenic properties of this drug is discussed.
Antibodies↗
Study of the sequence tagged site (STS) in the beginning of human apo A4 gene region.
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Apolipoproteins A↗
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.
7-Alkoxycoumarin O-Dealkylase↗
An avidin-biotin ELISA for the measurement of mitochondrial aspartate aminotransferase in human serum.
We describe a new double-antibody ELISA system using avidin-biotin amplication for the mass measurement of mitochondrial aspartate aminotransferase (m-AST) in human serum. The assay is very sensitive and as little as 0.5 ng/ml of m-AST may be detected. The method is linear up to 100 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 8.9% and 11.5% respectively for a low m-AST concentration (2.7 micrograms/l), and 5.9% and 8.0% respectively for a high level of m-AST (30 micrograms/l). The assay requires 100 microliters of serum and can be completed within 5 h. The ELISA procedure and a classical immunoprecipitation technique measuring the catalytic activity of the isoenzyme were applied simultaneously to the sera of 189 subjects. The protein levels determined by ELISA correlated poorly (r = 0.66) with the catalytic activity of m-AST.
Adolescent↗
An insertion deletion polymorphism in the signal peptide of the human apolipoprotein B gene.
In this communication we report the genetic properties of an insertion/deletion polymorphism in the signal peptide of the human apolipoprotein B (apo b) gene. There are two alleles of the apo B signal peptide; one codes for a peptide 27 amino acids in length and the other a peptide only 24 amino acids in length. Using the polymerase chain reaction the difference of nine nucleotides between the two alleles is readily detectable after electrophoresis of the amplification products. The relative frequencies of the Ins and Del alleles are 0.655 and 0.345, respectively. The apo B signal peptide genotypes are transmitted in a manner consistent with an autosomal codominant mode of inheritance with two alleles.
Alleles↗
Production, cross reactivity, and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase.
Eighteen IgG1 monoclonal antibodies (Mabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10(8) M-1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the Mabs (No 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other Mabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.
Animals↗
Development of an ELISA to study the polymorphism of dextromethorphan oxidation in a French population.
Simple, sensitive and selective enzyme immunoassays (ELISA) for monitoring urinary dextromethorphan and its major metabolite, dextrorphan, were developed. Dextromethorphan and dextrorphan hemisuccinates were linked to bovine serum albumin and specific antisera against each immunogen were raised in rabbits. The sensitivity of the ELISA was high (limit of detection 740 and 600 pg.ml-1 for dextromethorphan and dextrorphan, respectively). Intra- and interassay variation was less than 10%, and cross-reactivity between the two compounds was less than 1%. The ELISA was employed to phenotype 216 French subjects. The frequency of poor metabolizers was 5.1%.
Chromatography, High Pressure Liquid↗