[Molecular genetics: pre- and post-analytic "best practices"]. / Génétique moléculaire: les << bonnes pratiques>> pré-et postanalytieques.

Medical prescriptions for molecular genetic analyses are not yet very common in general practice, neverless they are becoming more and more frequent, and therefore it is more difficult to deal with them in part because of the recent french rules. Laboratory managers are supposed to be able to deal with such requests. This document, describing good laboratory practices, has been elaborated by members of the group "molecular genetics" from the "College National de Biochimie des Hôpitaux", providing details about: assessment of the prescriptions, including patient's consent, choice of the executing laboratory, specimen transmission, assessment and control of clinical and biological data, results transmission, confidentiality, archiving system. Such recommendations should facilitate exchanges with specialized laboratories, being specifically approved for practicing such analyses. The authors draw the attention of laboratory managers to the specificities of such requests.

Effect of ethyl esterification of phenolic acids on low-density lipoprotein oxidation.

Inhibition of copper-induced low-density lipoprotein (LDL) oxidation by phenolic acids and their ethyl esters was investigated. LDL oxidation was evaluated by the hydroperoxide concentration and the chromatographic pattern of apoprotein fractions after fast protein liquid chromatography (FPLC). Antiradical properties against 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) were also investigated, and lipophilicity determined by thin-layer chromatography. Caffeic acid at 5 microM and sinapic acid at 10 microM protected LDL against oxidation, inhibiting both hydroperoxide formation and the increase of apoprotein negative charge. Ferulic, gallic and p-hydroxy cinnamic acids were ineffective. Ethyl esterification increased the lipophilicity of the five acids, and enhanced the antioxidant properties of caffeic, sinapic and ferulic acids. Ethyl caffeate was protective at 1 microM. In contrast, gallic and p-hydroxy cinnamic ethyl esters were ineffective. Our results indicate that ethyl esterification of phenolic acids increases lipophilicity of their ethyl esters and may enable a better incorporation into the lipid layer of the LDL particle and the exertion of their antioxidant effect in the true site of lipoperoxidation. However, increasing lipophilicity is not the only mechanism able to potentiate preexisting antioxidant properties of molecules, and probably other mechanisms are implicated.

[Refinement and role of the diagnosis of Gilbert disease with molecular biology]. / Mise au point et intérêt du diagnostic de la maladie de Gilbert par biologie moléculaire.

Gilbert syndrome (GS), characterized by mild, chronic and isolated unconjugated hyperbilirubinemia is due to a partial deficiency of bilirubin-UDP-glucuronosyltransferase (UGT1A1). Recently, the genetic basis of GS has been identified in caucasian populations : it is related to the insertion of a dinucleotide (TA) in the promoter region of the UGT1A1 gene. In Asian populations, GS is due to missense mutations (either homozygous or heterozygous) in the coding sequence. The aim of this study was to develop a simple and rapid method to detect both genetic polymorphisms and mutations. This technique was performed (1) to explore unrelated unconjugated hyperbilirubinemia; (2) to evaluate the frequency of GS in a population of 97 healthy caucasian volunteers: 17% of them were homozygous for the TA7/TA7 polymorphism; (3) to determine the incidence of this syndrome in a population of 105 neonates with unconjugated hyperbilirubinemia. The incidence of GS (15%) was not significantly higher than it was in the control group. A correlation between GS genotype and neonatal jaundice was not established; (4) to seek a relationship between GS and preeclampsia with or without Hellp syndrome. The incidence in the Hellp syndrome group (n = 19) was 26%, two fold higher than in preeclampsia group (n = 22) and control group (n = 50) with only 14% and 13% respectively, (5) to start a study regarding the toxicity of irinotecan treatment in a population of homozygous children for the UGT1A1 polymorphism.

Genetic heterogeneity of glycogen storage disease type Ia in France: a study of 48 patients.

Forty-eight patients with glycogen storage disease type Ia (GSD Ia) were studied. Using a combination of single-strand conformation polymorphism (SSCP) analysis, restriction enzyme digestion and direct sequencing, we were able to identify 93/96 mutant alleles, comprising 23 different mutations in the glucose-6-phosphatase gene (G6PC). Among these, 7 are novel mutations of G6PC: M5R, T111I, A241T, C270R, F322L, and two deletions, 793delG and 872delC, resulting in the same mutation at the amino acid level, fs300Ter (300X).

In vitro study of the cytotoxicity of isolated oxidized lipid low-density lipoproteins fractions in human endothelial cells: relationship with the glutathione status and cell morphology.

Toxic effects of oxidized lipid compounds contained in oxidized LDL to endothelial cells are involved in the pathogenesis of atherosclerosis. Glutathione (GSH) plays an important role in the redox status of the cell and in the protective effect against oxidant injuries. However, little is known about the respective effect of these different oxidized lipid compounds toward cytotoxicity and GSH status of the cell. In this report, we isolated by high-performance liquid chromatography oxidized lipid compounds from low-density lipoproteins (LDL) oxidized by copper and we examined their effects on cultured endothelial cells. Cytotoxicity and GSH status were determined after incubation of endothelial cells with crude LDL or isolated lipid fractions derived from cholesterol, phospholipids, or cholesteryl esters. Their effects on cell morphology were also assessed. Oxidized lipids coming from cholesteryl esters (hydroperoxides or short-chain polar derivatives) induced a slight but significant GSH depletion without inducing cytotoxicity. The same species coming from phospholipids induced a more pronounced GSH depletion and a cytotoxic effect which is only present for the more polar compounds (short-chain polar derivatives) and corresponding to a total GSH depletion. In contrast, fractions containing oxysterols had a larger cytotoxic effect than their effect on GSH depletion suggesting that their cytotoxic effects are mediated by a GSH-independent pathway. All together, these data suggest that LDL-associated oxidized lipids present in copper-oxidized LDL exert cytotoxicity by an additional or synergistic effect on GSH depletion, but also by another mechanism independent of the redox status of the cell.

Implication of apolipoprotein E and the L-arginine-nitric oxide system in preeclampsia.

OBJECTIVE: During pregnancy, Apolipoprotein (Apo) E is synthesized in the placenta to facilitate the uptake of maternal lipoproteins. Preeclampsia is associated with an abnormal lipid profile. Apo E levels may affect the production of nitric oxide. We investigated whether Apo E variations could be related to the high lipid levels and nitric oxide secretion in preeclamptic women. METHODS: Blood samples from 15 normotensive women and 12 mild and 23 severe cases of preeclampsia were assayed for standard lipid profile, Apo E, and nitrate. Urine samples were analyzed for nitrate and cyclic GMP. RESULTS: In women with mild preeclampsia, the triglyceride concentration was significantly higher (p < 0.05) than in normotensive women (3.30 +/- 1.38 versus 2.31 +/- 0.92 g/L) and associated with a higher (p < 0.01) triglyceride/Apo E ratio (0.71; range = 0.40-1.70). In women with severe preeclampsia, the triglyceride/Apo E ratio was similar to normotensive women [0.39 (range = 0.18-1.19) versus 0.41 (range = 0.18-0.79)] associated with a normal triglyceride level and a twofold higher serum nitrate level [36 (range = 1-63 mumol/L) versus 14 (range = 1-37 mumol/L)]. CONCLUSION: The triglyceride/Apo E ratio is significantly higher in mild preeclampsia. In the severe form, this ratio is similar to that of normotensive pregnant women, probably due to a better uptake of triglyceride. Moreover, in the severe form, it is associated with a twofold normal serum nitrate level. Thus, Apo E and the nitric oxide status may be implicated in preeclampsia.

Kinetics of serum tumor marker concentrations and usefulness in clinical monitoring.

Only a few markers have been instrumental in the diagnosis of cancer. In contrast, tumor markers play a critical role in the monitoring of patients. The patient's clinical status and response to treatment can be evaluated rapidly using the tumor marker half-life (t(1/2)) and the tumor marker doubling time (DT). This report reviews the interest of determining these kinetic parameters for prostate-specific antigen, human chorionic gonadotropin, alpha-fetoprotein, carcinoembryonic antigen, cancer antigen (CA) 125, and CA 15-3. A rise in tumor markers (DT) is a yardstick with which benign diseases can be distinguished from metastatic disease, and the DT can be used to assess the efficacy of treatments. A decline in the tumor marker concentration (t(1/2)) is a predictor of possible residual disease if the timing of blood sampling is soon after therapy. The discrepancies in results obtained by different groups may be attributable to the multiplicity of immunoassays, the intrinsic characteristics of each marker (e.g., antigen specificity, molecular heterogeneity, and associated forms), individual factors (e.g., nonspecific increases and renal and hepatic diseases) and methods used to calculate kinetics (e.g., exponential models and timing of blood sampling). This kinetic approach could be of interest to optimize patient management.

Oxidized-LDL induce apoptosis in HUVEC but not in the endothelial cell line EA.hy 926.

We studied the cytotoxic effect of copper-oxidized LDL in human primary human umbilical vein endothelial cells (HUVEC) and the immortalized EA.hy 926 cell line. Copper oxidized LDL (50-200 microg apoB/ml) induced concentration-dependent apoptotic cell death in HUVEC but did not induce apoptosis in EA.hy 926 cells. Only necrotic EA.hy 926 cells were evidenced at all copper oxidized LDL concentrations (25-200 microg apoB/ml), oxidation states (lightly, moderately and extensively copper-oxidized LDL) and incubation periods (4, 8 and 20 h). The different mechanisms of cell death induced by copper-oxidized LDL in EA.hy 926 cells and HUVEC may be related to various factors such as cytokines. In this study, we investigated whether interleukin-8 may be implicated in this process. The interleukin-8 production was increased in EA.hy 926 cells but not in HUVEC incubated with oxidized LDL. This increase in EA.hy 926 cells was associated with necrosis but not apoptosis. Nevertheless, the addition of interleukin-8 to HUVEC did not inhibit apoptosis induced by oxidized LDL. As the lower antioxidant capacity of EA.hy 926 cells results in higher sensitivity to oxidized LDL cytotoxicity (as we previously described), the redox status of cells may also control the form of endothelial cell death. In atherosclerotic lesions, the formation of apoptotic endothelial cells may result in part from the induction by oxidized LDL.

Jaundice with hypertrophic pyloric stenosis as an early manifestation of Gilbert syndrome.

Jaundice associated with hypertrophic pyloric stenosis was recognised in three patients; previous reports have suggested that this is a possible early manifestation of Gilbert syndrome. Most patients with Gilbert syndrome are homozygous for a (TA)(7)TAA polymorphism in the gene promoter coding for bilirubin glucuronosyltransferase. Two of the reported patients were homozygous for the (TA)(7)TAA polymorphism whereas the third was heterozygous for the same polymorphism. Furthermore, no other factors contributing to jaundice in the three patients were found. These results suggest that jaundice associated with hypertrophic pyloric stenosis is due to molecular defects within the gene promoter.

Identification of three novel mutations (Q54P, W70X and T108I) in the glucose-6-phosphatase gene of patients with glycogen storage disease type Ia. Mutation in brief no. 256. Online.

Three novel mutations, Q54P, W70X and T1081, were identified in the gene encoding glucose-6-phosphatase in three patients with glycogen storage disease type Ia. Two sibs of Portuguese origin were homozygous for the Q54P mutation whereas the third patient, originating from both France and Lebanon, was a compound heterozygote for the W70X and T108I mutations. Glycogen storage disease type Ia is a heterogeneous autosomal recessive condition.

Prenatal diagnosis of glycogen storage disease type Ia by restriction enzyme digestion.

Glycogen storage disease type Ia (GSD Ia) is an autosomal recessive condition, caused by a deficiency of hepatic glucose-6-phosphatase (G6Pase) activity. In a consanguineous family originating from northern Africa whose first daughter was affected with GSD Ia, we were able to identify the disease-causing mutation, a cytosine to thymine substitution at nucleotide 326 in exon 2 of the G6Pase gene (R83C). This mutation causes the disappearance of an HgaI site, and is thus easily detectable by restriction enzyme digestion. Both parents were heterozygous for this mutation. During the third pregnancy, fetal genomic DNA was extracted from a chorionic villus biopsy sampled at the 24th week of gestation. Exons 2 of the G6Pase gene were amplified by the polymerase chain reaction followed by HgaI digestion. Fetal DNA analysis indicated that the fetus had received both normal G6Pase alleles. This result was confirmed after birth. DNA analysis is the only reliable method for prenatal diagnosis of GSD Ia.

[Glutathione peroxidases: value of their determination in clinical biology]. / Les glutathion peroxydases: intérêt de leur dosage en biologie clinique.

The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonstration of the consequences of the desequilibrium in the proxidant/antioxidant balance on biological molecules oxidation, intracellular signals transduction, apoptosis and necrosis, have led to new approach in the knowledge of many pathological processes. Methods for determining antioxidant capacity have been developed. The measurement of glutathione peroxidase activity is a key step in the study of oxidative stress. Its determination in clinical biology needs optimal conditions for standardised assays which will be used for epidemiological studies aimed to evaluate the role of nutritional factors involved in the pathogeny of diseases caused or accompanied by oxidative stress.

Comparison of oxidized low-density lipoprotein toxicity on EA.hy 926 cells and human vein endothelial cells: influence of antioxidant systems.

We compared the susceptibility to oxidized LDL cytotoxicity of primary human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells. EA.hy 926 endothelial cells were more susceptible than HUVEC. To determine the basis of this difference, we evaluated the enzymatic antioxidant machinery in the two cell types. The antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase were significantly lower in EA.hy cells than in HUVEC: 54%, 71% and 8% of the HUVEC enzyme activities respectively. Pre-incubation of the EA.hy 926 endothelial cells with glutathione peroxidase (100 IU/ml) inhibited the cytotoxic effect of oxidized LDL. Superoxide dismutase (300 or 600 IU/ml) and catalase (300 or 600 IU/ml) had no effect. Compared to HUVEC, the higher susceptibility of EA.hy 926 cells to oxidized LDL induced injury may be associated with lower antioxidant defences, in particular with lower glutathione peroxidase activity which is known to eliminate lipid hydroperoxides and thereby to prevent the formation of damaging peroxyl radical intermediates.

A method for simultaneous determination of plasma and erythrocyte antioxidant status. Evaluation of the antioxidant activity of vitamin E in healthy volunteers.

1. Since oxygen free radicals are directly involved in a variety of pathologies such as atherosclerosis, diabetes mellitus, inflammation and/or when a deficit of defences of the organism against radicals occurs, we developed a suitable and simple method to determine both the erythrocyte sensitivity to an oxidative stress and plasma antioxidant protective capacity. 2. This test is based on the introduction at 37 degrees C of a radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), within an erythrocyte suspension leading to a membrane alteration and ultimately to haemolysis. The latter can be quantified by determining the lacticodeshydrogenase activity released in the medium. The erythrocyte sensitivity to haemolysis and the volume of plasma inhibiting 50% of the haemolysis were determined. 3. Intra-assay CVs were 1.9% for erythrocyte sensitivity to oxidative stress and 3.4% for inhibitory 50% plasma volume. Inter-assay CVs for both erythrocyte sensitivity and inhibitory 50% plasma volume were 4%. 4. The reliability of this method was assessed and applied to test the protective effect of vitamin E, a well known antioxidant agent, in six healthy volunteers. Two weeks after daily administration of 500 mg of vitamin E, the mean plasma vitamin E concentration increased by 41% from 10.7 +/- 2.0 mg l-1 before treatment (P < 0.05). As the vitamin E concentration increased, the mean inhibitory 50% plasma volume and the percentage of haemolysed erythrocytes decreased respectively by 29% from 3.35 +/- 0.5 microliter (P < 0.05) and 18% from 71.5 +/- 3.8% (P < 0.05). No significative variation of these parameters was observed in six adult men without vitamin E supplementation. 5. Thus, this global and simple test permits an antioxidant status evaluation of a patient. It can be applied to various pathologies and allows the potency of new antioxidant molecules to be evaluated.

Oxidized low-density lipoprotein induces the production of interleukin-8 by endothelial cells.

The concentration of interleukin-8 (IL-8) and RANTES was measured in culture supernatants of human EA.hy 926 endothelial cells incubated with oxidized low-density lipoproteins (LDL). Oxidized LDL induced a 3-fold increase in IL-8 production (p < 0.01), whereas RANTES was not detected. Native LDL did not stimulate IL-8 production. IL-8 production in oxidized-LDL-treated cells was mediated by reactive oxygen species, as it was partially inhibited by catalase and completely inhibited by glutathione peroxidase and N-acetylcysteine (p < 0.01).

Decreased erythrocyte deformability in glycogen storage disease.

Liver glycogen storage diseases (GSD) are disorders associated with severe dyslipidaemia which can induce cell membrane alterations and possibly reduced cell deformability. Since decreased erythrocyte deformability is known to disturb blood flow in capillaries and may promote ischaemic diseases, this study was designed to investigate erythrocyte deformability using a new filtration system, the Cell Transit Analyser (CTA), and to examine lipid compounds in the blood of 23 patients affected with GSD, aged from 1 to 20 years and 18 controls aged from 1 to 17 years. The patients showed a mixed hyperlipidaemia with predominant hypertriglyceridaemia and an increase in erythrocytes mean transit times (TT) due to the presence of more rigid erythrocytes subpopulations when compared to controls. Thus the erythrocyte rigidity, in addition to the lipid abnormalities must be taken into account for long-term evolution of GSD patients. Moreover this cellular alteration may contribute to shortened erythrocyte survival.

Blood lipids and rheological modifications in glycogen storage disease.

OBJECTIVES: Hyperlipidemia is a feature of liver glycogen storage disease (GSD). Recent studies have suggested that rheological mechanisms such as elevated erythrocyte aggregation may be involved in the pathogenesis of ischemic syndromes associated with hyperlipidemia. DESIGN AND METHODS: We investigated erythrocyte aggregation, lipids, and circulatory proteins in the blood of 24 patients affected with GSD, aged from 1 to 23 years (mean = 8) and 26 controls aged from 1 to 28 years (mean = 9). RESULTS: The aggregation results were much higher in patients than controls. The lipid data showed a mixed hyperlipidemia with predominant hypertriglyceridemia, low HDL-C, apoA-I and LpA-I/A-II, and high apoB as compared with controls. However, the LpA-I was not significantly different from controls. CONCLUSIONS: In conclusion, patients with GSD presented hyperlipidemia and elevated erythrocyte aggregation such that they are at long-term risk of ischemic complications.

[Vitamin D and pubertal maturation. Value and tolerance of vitamin D supplementation during the winter season]. / Vitamine D et maturation pubertaire. Intérêt et tolérance d'une supplémentation vitaminique D en période hivernale.

BACKGROUND: In France, several cases of vitamin D-deficiency rickets among adolescents have been reported, but no prophylaxis measure has been systematically recommended at this age. The aim of this study was to measure 25-hydroxyvitamin D (25-(OH)D) levels and to search for biological signs of vitamin D deficiency during adolescence. Moreover, the effects of a unique oral dose of 100,000 IU of vitamin D3, given during the winter, were analysed. POPULATION AND METHODS: Circulating levels of 25-(OH)D, 1,25-dihydroxyvitamin D, intact parathyroid hormone (iPTH), alkaline phosphatase activities, calcium and phosphate were measured in 53 adolescents aged 10-17 years (81% of metropolitan origin), seen during the winter. The effect of a single oral dose of 100,000 IU of vitamin D3 or of placebo was studied in 15 of these subjects. RESULTS: 24.5% of the adolescents had low 25-(OH)D concentrations (< 6 ng/ml), this frequency being even more elevated (38%) at the end of pubertal maturation (stages 4 and 5). An increase in iPTH concentrations was found in subjects with lowest 25-(OH)D levels (< 3 ng/ml). An oral dose of 100,000 IU of vitamin D3 resulted in a significant increase in the 25-(OH)D levels; yet, these levels remained within the normal range during the 1-2 month follow-up of the nine treated subjects. This dosage made it possible to correct the low calcium concentrations (2.20-2.24 mmol/l) found before treatment in three adolescents. CONCLUSIONS: The high frequency of low vitamin D status observed during puberty and its normalization after a 100,000 IU vitamin D3 supplementation show the interest and safety of this intermittent 100,000 IU vitamin D3 supplementation to adolescents during the winter season.