Ultra high performance liquid chromatography-quadrupole-time of flight analysis for the identification and the determination of resveratrol and its metabolites in mouse plasma.

Resveratrol is a polyphenol that has numerous interesting biological properties, but, per os, it is quickly metabolized. Some of its metabolites are more concentrated than resveratrol, may have greater biological activities, and may act as a kind of store for resveratrol. Thus, to understand the biological impact of resveratrol on a physiological system, it is crucial to simultaneously analyze resveratrol and its metabolites in plasma. This study presents an analytical method based on UHPLC-Q-TOF mass spectrometry for the quantification of resveratrol and of its most common hydrophilic metabolites. The use of (13)C- and D-labeled standards specific to each molecule led to a linear calibration curve on a larger concentration range than described previously. The use of high resolution mass spectrometry in the full scan mode enabled simultaneous identification and quantification of some hydrophilic metabolites not previously described in mice. In addition, UHPLC separation, allowing run times lower than 10 min, can be used in studies that requiring analysis of many samples.

Catalase rs769214 SNP in elderly malnutrition and during renutrition: is glucagon to blame?

Impaired glucose tolerance is common during aging. The transcription factor PAX6 is involved in glucose homeostasis. Computational promoter sequence analysis of the catalase gene highlighted a putative PAX6 binding site on the rs769214 polymorphism A allele. Creation of this binding site has been suggested to explain renutrition inefficiency in malnourished elderly patients. Our aim was to evaluate the link between the rs769214 polymorphism of the catalase gene and glucose homeostasis in malnourished elderly patients at inclusion and during renutrition. Thirty-three malnourished elderly Caucasian inpatients were recruited. Nutritional and inflammatory statuses were assessed and a multiplex adipokine analysis was conducted at inclusion and discharge from the Geriatric Nutritional Care Unit at Charles-Foix Hospital (Ivry-sur-Seine, France). Serum glucagon, PAI-1, and TNF-α levels were significantly lower in the A-allele carriers at inclusion. During renutrition, A-allele carriers exhibited increased serum glucagon, PAI-1, and TNF-α variation. After renutrition, levels of these parameters were similar for A-allele carriers and G-allele carriers. A logistic ordinal multivariate regression analysis linked only variation of glucagon to rs769214 SNP. These results support a role for catalase SNP in the efficiency of renutrition in malnourished elderly patients via the modulation of glucagon secretion, probably involving PAX6.

Serum levels of adhesion molecules ICAM-1 and VCAM-1 and tissue inhibitor of metalloproteinases, TIMP-1, are elevated in patients with autoimmune thyroid disorders: relevance to vascular inflammation.

Serum levels of ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular cell Adhesion Molecule-1-I), TIMP-1 (tissue inhibitor of metalloproteinases 1) and MMP-9 (Metalloproteinase 9) are well established markers of inflammation. The physiopathological link between inflammation, atherosclerosis and autoimmunity is well demonstrated. However, serum levels of these biomarkers in patients with autoimmune-mediated dysthyroidism, including their evolution after improvement of the thyroid disorder have not been assessed. So, we evaluated the circulating levels of these markers in autoimmune and in non-autoimmune-mediated dysthyroid patients, and their evolution after treatment of thyroid disease. We conducted a prospective study to evaluate these markers before and after treatment in hyperthyroid patients (n = 33; 28 patients with autoimmune disease), hypothyroid patients (n = 38; 33 patients with autoimmune disease) and euthyroid subjects (n = 33). At baseline, serum levels of ICAM-1, VCAM-1 and TIMP-1 were significantly elevated in patients with hyperthyroidism as compared to euthyroid and hypothyroid patients (respectively p = 0.0005 and p < 0.0001). In multivariate analysis, the differences remained significant for VCAM-1 and TIMP-1. Median levels of ICAM-1, VCAM-1 and TIMP-1 were significantly higher in patients with autoimmune-mediated dysthyroidism compared to euthyroid patients (respectively p < 0.0001 and p = 0.002). In hyperthyroid patients, ICAM-1, VCAM-1 and TIMP-1 concentrations fell significantly after they had become euthyroid (respectively p = 0.0006; p < 0.0001 and p = 0.0009), although VCAM-1 values remained higher than those observed in the control group (p = 0.005). We found that autoimmune-mediated dysthyroidism were associated with increased peripheral blood concentrations of VCAM-1, ICAM-1 and TIMP-1. Whether these biological abnormalities translate into increase intima remodelling and atherosclerosis remains to be studied.

[Prognostic value of S-100B protein plasma measurement after cardiac arrest]. / Intérêt pronostique du dosage de la protéine S-100B sérique au décours d'un arrêt cardiaque en milieu extrahospitalier : données préliminaires françaises.

S-100B protein is selectively synthesized by glial cells, and is released in biological fluids after acute brain damage. We analyzed initial levels and evolution of plasma S-100B protein concentrations after resuscitated cardiopulmonary arrest (CPA). S-100B levels were determined in 27 subjects at the time of CPA (H0) then 12, 24 and 48 h after resuscitation. Initial levels of S-100B and kinetics revealed that: 1) 95% the of subjects with a concentration of protein S-100B greater than 0.80 microg/L at H0 did not survive; 2) 62% of subjects with a concentration of protein S-100B lower than 0.80 microg/L at H0 survived; 3) 100% of subjects with a protein S-100B level lower than 0.80 microg/L at H0 and whose evolution kinetics of S-100B levels showed a decrease survived; 4) 100% of the subjects whose S-100B levels increased from H12 died. In summary, this study suggests that the threshold of 0.80 microg/L for S-100B plasma levels at H0 could be predictive for the outcome of the CPA, when associated with the kinetic study of S-100B plasma concentration.

[Preanalytical guidelines for clinical proteomics investigation of biological fluids]. / Recommandations préanalytiques pour les analyses de protéomique clinique des fluides biologiques.

Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.

["Standard" protocol for evaluating the impact of preanalytical variables on peptidic and proteic analytes and standard coding of preanalytical procedures]. / Procédure << standard >> pour tester l'impact des variables préanalytiques sur des analyses peptidiques et protéiques et proposition de codage << standard >> des procédures préanalytiques.

The SFBC Working Group on << Preanalytics and multiplex analyses in proteomics >> is presenting a protocol which will allow harmonization of biospecimen research studies on the impact of different preanalytical variations on peptidic and protein analytes. This protocol is based upon standardization of preanalytical options corresponding to different preanalytical variations and different types of biospecimens (serum, plasma, cedrebrospinal fluid and urine). Application of this protocol will allow, not only harmonization of Biospecimen research, but also elaboration of standard nomenclature of the preanalytical steps.

[Interference of hemoglobin variants on glycated hemoglobin measurement using Architect ci8200 immunoassay (Abbott Diagnostics)]. / Immunodosage de l'hémoglobine glyquée sur ci8200 (Abbott Diagnostics) en présence d'hémoglobines anormales.

The objective of this study was to compare haemoglobin A(1c) (HbA(1c)) levels measured by the immunoturbidimetric assay on ci8200 Architect (Abbott Diagnostics) to those obtained by the high pressure liquid chromatography (HPLC) assay from Tosoh Bioscience. Firstly, we verified correlation between the two methods in 47 subjects without hemoglobin variants: the coefficient of correlation was 0.968 and the regression equation was as follows: y(Abbott) = 0.928x(Tosoh) - 0.081. We then measured HbA(1c) levels by both methods in blood samples of 23 patients with a structural hemoglobin variant (A/S, A/C or S/C). Analysis of these samples revealed significant discrepancies between results of both methods, as indicated by the higher mean value obtained by immunoturbidimetric assay when compared to HPLC assay (7.17 +/- 2.68% vs. 5.86 +/- 1.41%). Classification of patients according to the HAS (French Haute Autorité de Santé) recommendations HbA(1c) was not modified for 68% of patients with abnormal hemoglobin. However, in 32% of cases, discrepancies between the two methods may have clinical importance, which justifies the occurrence of an abnormal hemoglobin when the HbA(1c) assay is performed by the method immunoturbidimetry Abbott Diagnostics.

[Multiplexed analysis for identification and evaluation of novel biomarkers in biological fluids, tissue and cell extracts]. / Méthodes d'analyses moléculaires multiplexes sur supports solides ou en milieu liquide pour l'identification de biomarqueurs protéiques dans les fluides biologiques et les extraits cellulaires ou tissulaires.

This manuscript reports the conclusions of a working group of the French Society of Clinical Biology (SFBC) 2007-2008 which evaluated the status, the impact and the future developments related to the analysis of the proteome using multiplexed methods. These approaches that are also used in clinical biology are performed on solid supports or in flow by using specifically dedicated or conventional flow cytometers. This review provides therefore a broad overview going from the methods already present in research laboratories to the tools for clinical and medical investigations.

[Recommendations for metrological control of measuring equipments in the clinical laboratories]. / Recommandations pour la maîtrise métrologique des équipements de mesure au laboratoire d'analyses de biologie médicale (Document D).

The control of the measuring equipment (balances, mass standards, micropipettes, dilutors, volumetric glassware, thermometers...) is essential to obtain reliable analytical results. This control requires knowledge and use of the main standards, instructions for use, metrological verifications and confirmations as well as creation of checking and standardizing certificates ensuring metrological traceability. These items should help to control the quality of the analytical performances (fidelity and trueness in particular).

[S100B protein: a novel biomarker for the diagnosis of head injury]. / La protéine S100B: premier marqueur biologique pour le diagnostic du traumatisme crânien mineur ou modéré.

Acute head injury is a common reason for seeking care in an emergency department and in France accounts for more than 500 consultations daily. In most cases, though the trauma is minor/moderate, a computed tomography scan is necessary to establish the diagnosis and avoid medical and/or social sequelae. This examination is however expensive and not always readily available in the emergency setting. Until recently clinical chemists were unable to propose to physicians a pertinent biomarker of acute brain injury. S100B protein is a constitutive protein of glial cells, whose physiological functions are both intracellular, i.e. intracytosolic calcium binding, and extracellular, e.g. by promoting neuritic proliferation and/or neuronal apoptosis. Due to specificity of its cellular expression, S100B protein is a useful biological marker of acute neurological disorders, such as ischemic or hemorrhagic stroke, and traumatic brain injury. This brief review exposes the contribution of S100B measurement in biological fluids to the diagnosis, the follow-up, and the prognosis of acute minor/moderate head injury. In addition, we describe the French STIC-S100 study, a cohort study designed to determine the negative prognostic value of early determination of plasma S100B levels for the diagnosis of minor/moderate head injury and its medical/social consequences.

[Biochemical exploration of energetic metabolism and oxidative stress in low grade gliomas: central and peripheral tumor tissue analysis]. / Exploration biochimique du métabolisme énergétique et du stress oxydant des gliomes de bas grade: analyse du tissu tumoral central et périphérique.

Gliomas represent 50% of primary brain tumors, and their prognosis remains poor despite the advances in diagnosis and therapeutic strategies. Low grade gliomas (LGG) are infiltrative tumors and they constantly undergo malignant transformation. Metabolic exploration of human gliomas in vivo, in animals and by using cell culture models showed important differences between tumor tissues and normal brain tissues, which can provide new markers for diagnosis, prognosis and therapeutic targets. In this study, energetic and oxidant metabolisms were explored in biopsy extracts of LGG obtained from the centre and the periphery of tumors. Metabolic pattern of these tumors was explored and the differences between the centre and the periphery pointed. Our study showed a metabolic heterogeneity between tumors, with hypermetabolic and hypometabolic profiles. Lactate to pyruvate ratio was>1, suggesting that the energy metabolism in LGG is glycolytic in nature, particularly in the centre of the tumors. Peripheral samples of tumors showed increased glucose consumption and cytochrome c oxidase activity. Lipid peroxidation and catalase activity were also increased in the periphery compared to the centre of tumors. A relationship between the main antioxidant and energy metabolism enzymes activities was observed, suggesting that periphery of tumors is more active metabolically and more resistant to free radical injury.

[Cellular energetic metabolism of cerebral tissue: metabolic characteristics of glial tumours]. / Métabolisme énergétique cellulaire du tissu cérébral: spécificités métaboliques des tumeurs gliales.

This review reports recent observations concerning specificities of the cellular energy metabolism in cerebral tissues that highlight on characteristics of that of glial tumours, such as the association of metabolic alterations aggressiveness of these tumours. Compared to normal cerebral tissue, glial tissue exhibits both a relative independence towards oxygen and substrate furnitures and thus vascularization, as well as the metabolic co-operation of neurons and glial cells within the tumour. Occurrence of a Warburg effect could explain such metabolic autonomy that might be associated to genetic changes observed in gliomas. Characteristics of the glycolytic metabolism within glioma tissue therefore may be novel land therapeutic approaches for the treatment of these tumours.

[Present possibilities and future development of clinical proteomics]. / Présent et futur de la Protéomique clinique.

This review focuses on "clinical proteomics" which represents an emerging discipline in biomedical research. "Clinical proteomics" relies on the analysis of the proteome, i.e. the entire set of peptides and proteins present in a biological sample, to provide relevant data for diagnosis, prognosis or therapeutic strategies of human pathologies. This new type of approach has tremendous potential for the diagnosis of complex pathologies or for the early detection of cancers. This article reports the conclusions of a workgroup of the French Society for Clinical Biology (SFBC) 2004-2006 which evaluated the status, the impact and the future development of proteomics in the clinical field. It provides therefore a broad view going from the methods already present in the clinical laboratories (multiplex technologies...), to the tools for clinical and basis research including bioinformatics.

Cytoprotective effect against UV-induced DNA damage and oxidative stress: role of new biological UV filter.

The majority of chemical solar filters are cytotoxic, particularly on sensitive ocular cells (corneal and conjunctival cells). Consequently, a non-cytotoxic UV filter would be interesting in dermatology, but more especially in ophthalmology. In fact, light damage to the eye can be avoided thanks to a very efficient ocular antioxidant system; indeed, the chromophores absorb light and dissipate its energy. After middle age, a decrease in the production of antioxidants and antioxidative enzymes appears with accumulation of endogenous molecules that are phototoxic. UV radiations can induce reactive oxygen species formation, leading to various ocular diseases. Because most UV filters are cytotoxic for the eye, we investigated the anti-UV properties of Calophyllum inophyllum oil in order to propose it as a potential vehicle, free of toxicity, with a natural UV filter action in ophthalmic formulation. Calophyllum inophyllum oil, even at low concentration (1/10,000, v/v), exhibited significant UV absorption properties (maximum at 300nm) and was associated with an important sun protection factor (18-22). Oil concentrations up to 1% were not cytotoxic on human conjunctival epithelial cells, and Calophyllum inophyllum oil appeared to act as a cytoprotective agent against oxidative stress and DNA damage (85% of the DNA damage induced by UV radiations were inhibited with 1% Calophyllum oil) and did not induce in vivo ocular irritation (Draize test on New Zealand rabbits). Calophyllum inophyllum oil thus exhibited antioxidant and cytoprotective properties, and therefore might serve, for the first time, as a natural UV filter in ophthalmic preparations.

[Cellular sources of reactive oxygen and nitrogen species. Roles in signal transcription pathways]. / Sources cellulaires des espèces réactives de l'oxygène et de l'azote. Implication dans la transcription et la régulation des gènes.

The history of studies regarding reactive oxygen and nitrogen species (ROS/RNS) is approximatively of 50 years. ROS were shown initially for their deleterious effects on marcormolecules such as DNA and proteins, leading to deterioration of cellular functions as an oxidative stress. On the other hand, recent studies have demonstrated that ROS/RNS act as oxidative signalling in cells, resulting in various gene expressions. This brief review focuses on the main cellular origins of ERO/ERN, such as mitochondrial respiratory chain, NAD(P)H oxidase and NO synthases, and describe the modulation by the reactive species of two major signal transduction pathways, NF-KB and AP-1 pathways.

Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endothelial cells through the inhibition of protein kinase C.

Oxidative stress plays a major role in the pathogenesis and in the onset of macrovascular complications of diabetes. We previously reported that the antihyperglycaemic drug metformin was able to decrease significantly intracellular reactive oxygen species (ROS) production of bovine aortic endothelial cells (BAEC) activated by high levels of glucose and angiotensin II (ANG). The aim of the present study was to investigate whether the antioxidant effect of metformin on BAEC could be mediated through a modulation of protein kinase C (PKC) activity, which plays a key role in the pathophysiology of diabetes. The effects of metformin on intracellular ROS production, PKC translocation and activity were studied on endothelial cells stimulated by PMA (a direct PKC activator), ANG or high levels of glucose as pathophysiological stimuli of endothelial dysfunction in diabetes. We showed that metformin decreased ROS production on PMA-, ANG- and glucose-stimulated BAEC in a similar manner to that obtained by PKC specific inhibitors (calphostin C, chelerythrine) alone. On the other hand, metformin reduced both PKC membrane translocation and kinase activity in ANG-stimulated cells. In PMA-activated cells, metformin reduced membrane PKC activity but we did not observe any alteration of PKC membrane translocation. Finally, in vitro incubation with purified PKC indicated that metformin had no direct effect on PKC activity. Taken together, our results suggest that metformin exerted intracellular antioxidant properties by decreasing ROS production through the inhibition of PKC activity.

Elevated circulating levels of matrix metalloproteinase-9 in type 1 diabetic patients with and without retinopathy.

OBJECTIVE: Tissue expression pattern of matrix metalloproteinases (MMPs) and their inhibitors TIMPs indicate that microvascular complications of diabetes mellitus are associated with extracellular matrix remodelling. We investigated whether circulating levels of MMP-9 and TIMP-1 are altered in diabetic retinopathy and whether they might serve as biological markers of ocular complications in type 1 diabetes. RESEARCH DESIGN AND METHODS: We recruited 47 type 1 diabetic patients free of vascular complications (n=40) or with retinopathy (n=14). Patients with macroangiopathy, neuropathy and nephropathy were excluded. A group of nondiabetic control subjects (n=35) was also constituted for comparative purposes. Peripheral blood levels of MMP-9 and TIMP-1 were determined using immunoenzymatic assays. RESULTS: Type 1 diabetic subjects exhibited significantly higher circulating levels of both MMP-9 and MMP-9/TIMP-1 ratio, as well as a tendency to increased serum TIMP-1 levels relative to nondiabetic controls (p<0.001). Diabetic patients with retinopathy also displayed elevated systemic values of MMP-9 and MMP-9/TIMP-1 ratio when compared to patients without retinopathy (p<0.05). Logistic regression analysis identified diabetes duration firstly (P<0.01), and MMP-9 serum levels secondly (P<0.01) as significant and independent variables associated with the existence of retinopathy. CONCLUSIONS: Our data suggest that peripheral blood MMP-9 levels might serve as surrogate biomarkers of retinopathy in type 1 diabetic patients free of other vascular complications.

[Sodium/iodide symporter: physiopathological aspects and therapeutic perspectives]. / Le symporteur sodium/iodure: données récentes et perspectives thérapeutiques.

The existence of the natriuric/iodide symporter (NIS) represents a new view to understand the thyroid metabolism of iodide. Due to its cellular localisation on basolateral membrane, this transporter exerts an essential role in the biological functions of the thyroid, especially the capacity to accumulate iodide into the thyrocytes. Clinical perspectives of NIS activity modulation would ameliorate the diagnosis and the treatment of thyroid diseases by using radioisotopes transported by the NIS (131 iodide, 99m technetium, 188 rhenium). The study of the regulation pathways modulating the expression and the activity of the symporter NIS, would allow to understand pathogeny of benign or malignant diseases of the thyroid gland. The relative facility of the therapy management by 131 iodide and its good efficiency associated to the recent advance of NIS function also give an interesting perspective to the gene therapy treatment of the nonthyroid cancers despite existent methodological problems.

[Analytical aspects of the semiquantitative determination of urinary iodine using ferroin: value of rapid screening for iodine deficiency or excess]. / Aspects méthodologiques de la détermination semi-quantitative de l'iodurie par la ferroïne: intérêt dans le dépistage rapide de carence ou surcharge en iode.

Iodine is an essential element for thyroid hormone synthesis. Iodine disorders induced biological and/to clinical expression of thyroid dysfunction. Inappropriate iodine intake (by default or by excess) is worrying in terms of public health in France regarding the iodine deficiency and the frequency of iatrogen iodine overloads. Urinary iodine determination which generally implicates the use of a cerimetric method, is an useful tool to evaluate iodine intakes. In this study, we described the analytical aspects of a semiquantitative method of urinary iodine using a redox indicator, ferroin. This method allows the screening of iodine excess or deficiency in a short time (< 3 hours) with a good specificity and sensitivity. Since this assay does not require specific apparatus, it could be easily developed in clinical chemistry laboratories for the detection of inappropriate iodine intakes, and could be useful for prevention programs of iodine deficiency.