[Reproductibility of the morphological identification of schisocytes and evaluation of non observer-dependent methods]. / Schizocytes : reproductibilité du diagnostic morphologique et intérêt de méthodes observateur-indépendantes.

Schistocytes are red blood cell fragments observed on a blood smear. They are a mark of mechanical haemolytic anaemias whose group of the thrombotic micro-angiopathies requires an urgent treatment. The detection of the schistocytes and sometimes their quantification are thus of primary importance. To evaluate this search for schistocytes, several surveys of practice were carried out (1999-2003) including pictures of blood fields (identification of schistocytes among abnormal red blood cells) near biologists of variable level of specialization. The aim was to try to lead to a consensus, in particular for the morphological criterias of identification. Our results indicated that: 1) the biologists are badly sensitized with the importance of this research and the consequences of their response for the diagnosis; 2) the morphological identification of the schistocytes is difficult with an important variability of the criteria according to the observers. An investigation overviewed by the French Group of Cellular Hematology (Delphi method) allowed the development of a morphological consensus (fragments of triangular/crescent/helmet forms with rectilinear zone testifying to the zone of break). In order to cancel the observer-dependent identification of the schistocytes, a software of morphometric analysis (Q-WIN, Leica) was developed for sorting, starting from digitalized microscopic fields, the fragmented - among the normal - red blood cells. The results appeared encouraging, but not yet optimized. An automated analyzer (Bayer ADVIA 120) was also evaluated for the measurement of the schizocytes ("fragmented red blood cells" parameter). The moderate over-estimation of the real schizocytes (+ 0,4%) encouraged to observe the clinical value of the fragmented red blood cells detection in a group of patients that undergone a bone-marrow transplantation. The predictive value of the test (98%) was satisfactory.

Novel mutations of CYP3A4 in Chinese.

Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence of CYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A(17776), designated as CYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6beta-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6beta-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4, CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic groups and its effect on the metabolic activity of CYP3A4 remain to be further evaluated.

Avidin-biotin enzyme immunoassay of osteocalcin in serum or plasma.

We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs.

The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3'-azido-3'-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mM) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent Km and Vmax revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest.

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%.

[Variation in the urinary excretion of 6-beta-hydroxycortisol in humans after administration of the new isoquinoline derivative, PK-11195 (52028 RP)]. / Variation chez l'homme de l'excrétion du 6-beta-hydroxycortisol urinaire après administration d'un nouveau dérivé de l'isoquinoléine, PK-11195 (52028 RP).

Among in vivo tests to assess liver drug metabolizing enzyme induction, urinary 6-beta-hydroxycortisol (6-beta-OHF), plasma gamma-glutamyltransferase (GGT) and urinary D-glucaric acid are most frequently proposed. 6-beta-OHF is the most abundant unconjugated metabolite of cortisol in human urine. We measured its elimination during a clinical trial in 16 human healthy volunteers (men and women), these persons being treated by a new isoquinoleine derivative, 52028 RP (PK-11195). This drug is an antagonist of peripheral type benzodiazepine binding sites. Urinary excretion of 6-beta-OHF increased significantly (3.5 fold, p less than 0.01) on the 5th day of treatment (400 mg/day, orally) and remained increased as long as the treatment was continued (15 days). Control values were again observed 5 days after stopping the treatment. Plasma gamma-glutamyltransferase activity and D-glucaric acid urinary elimination are increased more than 2 fold. The data demonstrated that 6-beta-OHF in the most sensitive among the three tests performed to detect a drug metabolism induction, during this clinical trial.