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Introduction The quantitation of glucagon remains a challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work. Methods Glucagon was extracted from plasma employing a mixed-mode anion exchange solid-phase extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories. Results Assay imprecision was below 4%. Recoveries were within 95–103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4 °C for less than 4 h. Diasource® RIA disagreed with our method. Mercodia® ELISA provided a closer agreement, also proven by external quality control samples. Conclusions A rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon.

Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4–27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were 21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.

Introduction Determination of creatinine and estimation of Glomerular Filtration Rate (GFR) rapidly before injection of contrast media provides early detection of high-risk patients for acute kidney failure. Hence, a rapid point-of-care (POC) device (result in 30 s) allowing creatinine measurement and eGFR could be of interest. To validate this method, we considered a population referred for measuring GFR. Methods Iohexol plasma clearance was used to measure GFR. For each subject, enzymatic creatinine was quantified with two different devices: in plasma with the Roche Cobas analyzer and in capillary blood with the Nova Biomedical POC device. Both values of creatinine were used in the CKD-EPI equation for estimated glomerular filtration rate (eGFR). eGFR using POC was compared to eGFR using Cobas and to mGFR by Passing Bablok regression, calculation of bias, precision and accuracy (or concordance) within 30%. Also, we calculated the rate of discrepant staging (eGFR >60 or ≤ 60 when mGFR is actually ≤60 and > 60) with both creatinine methods. Results 120 subjects (52 ± 13 years, 49% of women) were included. Mean mGFR was 77 ± 27 mL/min/1.73m2 with 29 patients presenting mGFR 60 mL/min/1.73m2 whereas mGFR it was ≤60 and 10% (9.2%) of them would have been considered with GFR ≤60 mL/min/1.73m2 when mGFR was >60. Conclusion Creatinine measured with the POC has an acceptable performance when used with the CKD-EPI equation to estimate GFR. Its ability to detect GFR

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 25-hydroxyvitamin D3/D2 (25(OH)D3/D2) in human aqueous humor samples from the Eye-D study. Optimum sample preparation is based on simple liquid–liquid extraction (LLE) followed by derivatization with an Amplifex reagent prior to LC-MS/MS in order to enhance analyte sensitivity. The average recoveries for 24,25(OH)2D3 (0.02, 0.05, 0.4 μg L−1), 25(OH)D3 (0.2, 0.5,3.75 μg L−1) and 25(OH)D2 (0.15, 0.4, 3 μg L−1) ranged from 92 to 112 with a coefficient of variance (CV) lower than 15%. The limits of quantitation (LOQs) were from 0.02 μg L−1 (24,25(OH)2D3) to 0.2 μg L−1 (25(OH)D3) using a sample volume of 50 μL. We demonstrated that the present method provides adequate sensitivity, selectivity, accuracy, and robustness to screen for vitamin D metabolites in aqueous humor samples by analyzing five samples which were withdrawn during cataract operation.

Background: A key role of oxidative stress has been highlighted in the pathogenesis of COVID-19. However, little has been said about oxidative stress status (OSS) of COVID-19 patients hospitalized in intensive care unit (ICU). Material and Methods: Biomarkers of the systemic OSS included antioxidants (9 assays), trace elements (3 assays), inflammation markers (4 assays) and oxidative damage to lipids (3 assays). Results: Blood samples were drawn after 9 (7–11) and 41 (39–43) days of ICU stay, respectively in 3 and 6 patients. Vitamin C, thiol proteins, reduced glutathione, γ-tocopherol, β-carotene and PAOT® score were significantly decreased compared to laboratory reference values. Selenium concentration was at the limit of the lower reference value. By contrast, the copper/zinc ratio (as a source of oxidative stress) was higher than reference values in 55% of patients while copper was significantly correlated with lipid peroxides (r = 0.95, p &lt, 0.001). Inflammatory biomarkers (C-reactive protein and myeloperoxidase) were significantly increased when compared to normals. Conclusions: The systemic OSS was strongly altered in critically ill COVID-19 patients as evidenced by increased lipid peroxidation but also by deficits in some antioxidants (vitamin C, glutathione, thiol proteins) and trace elements (selenium).

An intralaboratory study assessing assay variability and bias for determination of serum total 25-hydroxyvitamin D [25(OH)D] was conducted by the Vitamin D Standardization Program (VDSP). Thirteen assays for serum total 25(OH)D were evaluated in a single laboratory including 11 unique immunoassays and one liquid chromatography – tandem mass spectrometry (LC-MS/MS) assay. Fifty single-donor serum samples, including eight samples with high concentrations of 25(OH)D(2) (> 30 nmol/L), were assigned target values for 25(OH)D(2) and 25(OH)D(3) using reference measurement procedures (RMP). Using four replicate measurements for each sample, the mean total percent coefficient of variation (%CV) and mean % bias from the target values were determined for each assay using the 50 single-donor samples and a 42-sample subset, which excluded 8 high 25(OH)D(2) concentration samples, and compared with VDSP performance criteria of ≤ 10% CV and ≤ ±5% mean bias. All 12 assays achieved the performance criterion for %CV, and 9 of the 12 assays were within ≤ ±5 % mean bias. The Fujirebio Inc. assay exhibited the lowest %CV and highest percentage of individual measurements within ≤ ±5% mean bias. Ten immunoassays exhibited changes in response due to the high 25(OH)D(2) samples with Abbott, Biomérieux, DiaSorin, DIAsource, and IDS-iSYS assays having the largest deviations. The Fujirebio Inc. and Beckman Coulter assays were only minimally affected by the presence of the high 25(OH)D(2) samples. Samples with high concentrations of 25(OH)D(2) provided a critical performance test for immunoassays indicating that some assays may not have equal response or recovery for 25(OH)D(2) and 25(OH)D(3).

Steroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species. The sample preparation procedure is based on a simple protein precipitation and a derivatization with dansyl chloride. After the chromatographical separation, compounds were analyzed with a triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Mare and American bison serum samples were analyzed with the validated method and results were compared with concentrations measured with commercial radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA). Following these criterions: relative standard deviation15% and relative bias15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison between immunoassays and LC-MS showed poor correlation and proportional differences. Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool for veterinary clinicians and LC-MS should thus be used to update and refine the current knowledge about the endocrinology of pregnancy in mammals.

Vitamin D status is typically assessed by the measurement of 25-hydroxyvitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, which was identified with a synthesized pure substance. Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analyzed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100 A core-shell column (150 × 4.6 mm) coupled to a Sciex 4500 mass spectrometer. For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were

© 2018 Background: Aldosterone measurement is critical for diagnosis of primary aldosteronism and disorders of the renin-angiotensin system. We developed an LC-MS/MS method for plasma and urinary aldosterone and compared it to our RIA method. We present a reference interval study for a Belgian population. Methods: 68 plasma and 23 urine samples were assayed for as part of a method comparison. For the reference interval study, we enrolled 282 healthy Caucasian volunteers (114 Male: mean age 35 ± 11 y and 168 Female: mean age 42 ± 13 y). A subset of 139 healthy volunteers agreed to a 24-h urine collection. For the method validation, 5 plasma and 8 urine pools were run in triplicate and quadruplicate, respectively, on 3 different days. Results: Between-run imprecision (CV) was 2.8–5.1% for plasma and 4.5–8.6% for urine, except at the low urine concentration of 2.99 nmol/L where a CV of 15.4% was observed. The limit of quantitation was 0.04 nmol/L for plasma and 6.65 nmol/L for urine. Recoveries, based on spiking experiments into natural matrix, did not differ significantly from 100%. Regression comparisons showed that, on average, RIA generated results were 59% and 11% higher than LC-MS/MS for plasma and urine, respectively. The MS reference interval we propose for plasma aldosterone is 0.07 nmol/L–0.73 nmol/L for women and 0.04 nmol/L–0.41 nmol/L for men. No gender difference was observed for urine aldosterone. The reference interval was determined to be

Background Burn patients are at risk of vitamin D (VD) deficiency and may benefit from its pleiotropic effects as soon as acute phase. Aim of this observational study was to assess effects of a cholecalciferol (VD3) bolus on VD status in adult burn patients (Group B, GB) after admission, compared to healthy subjects (Group H, GH). Methods Both groups received an oral dose of 100,000 IU VD3. Blood samples were collected before (D0) and 7 days (D7) after bolus to measure 250H-D, 1,25(OH)2-D, parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). Albumin (ALB) and VD binding protein (DBP) were measured and used to calculate free 25OH-D level. Data were expressed as median (min–max) or proportions. Results A total of 49 subjects were included: 29 in GH and 20 in GB. At D0, prevalence of VD deficiency was higher in GB: 25OH-D was 21.5 (10.1–46.3) ng/ml in GH vs 11 (1.8–31.4) ng/ml in GB. DBP and ALB were lower in GB. At D7, DBP was stable in both groups while ALB decreased in GB. 25OH-D increased by 66.6 (13.5–260.3)% in GH. In GB, changes in 25OH-D extended from −36.7% to 333.3% with a median increase of 33.1%. Similar changes were observed in each group for free 25OH-D. High FGF23 levels were observed in GB. Conclusions This study highlighted the differences in VD status and in response to a high dose VD3 in burn patients when compared to healthy patients. Pitfalls in VD status assessment are numerous during acute burn care: 25OH-D measurement needs cautious interpretation and interest of free 25OH-D is still questionable. They should not prevent burn patients to receive VD supplements during acute care. Higher doses than general recommendations should probably be considered.

Background:We provide a clinical and analytical evaluation of the reformulated version of the Abbott Architect 25-hydroxyvitamin D assay. We compared this assay with three commercial automated immunoassays and against a VDSP-traceable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in six different populations. We also supplemented 40 healthy volunteers with either 600,000 IU of vitamin D2 or 100,000 of vitamin D3 to evaluate the performance of the immunoassays vs. the LC-MS/MS.Methods:Precision and limit of quantification were assessed, 25(OH)D2 and C3-epimer recovery were calculated. Two hundred and forty samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and LC-MS/MS. We verified if patients were homogeneously classified with the immunoassays when they took vitamin D2 or vitamin D3 after 1, 7 and 28 days.Results:We observed excellent analytical features and showed a very good correlation to the LC-MS/MS results in the overall population. Compared to the other immunoassays, concordance of the new Abbott assay with the LC-MS/MS was at least similar, and often better in diseased populations. Althought the cross-reactivity with 25(OH)D2 was not of 100%, there was no significant difference in the classifications of the patients, either supplemented with D2 or D3 or after 7 or 28 days.Conclusions:This modified version of the Abbott Architect assay is clearly improved compared to the previous one and presents a better agreement with the LC-MS/MS.

Background Interpretation of 25OH-D measurement during critical care (CC) may be problematic due to variations of binding protein concentrations (albumin, ALB, and vitamin D binding protein, VDBP). Determination of free 25OH-D concentration may thus be relevant in CC patients. The aim of this observational study was to evaluate effects of an acute hemodilution on vitamin D (VD) status. Methods Blood samples were obtained before (T1) and after a crystalloid load (T2) administered at anesthesia induction for minor surgery. 25OH-D was measured with LC–MS/MS and with 3 immunoassays (IA): DiaSorin Liaison, IDS iSYS and bioMerieux Vidas. VDBP was measured with the R&D Elisa and ALB on Cobas. Free 25OH-D was calculated using published formula. Accuracy of each 25OH-D IA was calculated as the percentage of IA values within 20% of their respective LC–MS/MS values. Performances of the three AI were compared with LC–MC/MS using Bland–Altman analysis. Results Twenty adults were included. Compared to T1 values, VDBP, ALB and LC–MS/MS values decreased in parallel by a mean of 23% at T2. IA values decreased less significantly (12, 14 and 15% for Liaison, iSYS and Vidas, respectively). IA-based calculated free 25OH-D significantly increased after dilution, while LC–MS/MS-based free values remained stable. At T1 and T2, bias were demonstrable for all IA. After hemodilution, bias would lead to overestimation for the three IA. Accuracy of IA decreased after dilution. Conclusions Due to matrix effects, compared to LC–MS/MS, IA results were impacted by hemodilution. In CC patients, LC–MS/MS seems to be the best option to measure 25OH-D. Specific LC–MS/MS method should be developed to measure free 25OH-D.

Introduction: A fatty acid (FA) is a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Recently, the role of FA and particularly omega-3 and -6 has emerged as cardiovascular risk factor in the literature. The aim of our study was to establish reference values for these FA and to compare them with data obtained in a population of acute myocardial infarction (AMI) patients. Materials and methods: Hundred thirty five healthy subjects (59.38 ± 27.12 yo, 75 men) were selected as reference population. We also evaluated FA in thirty three patients (55 ± 9 yo, 23 men) admitted in the Emergency Department of our Institution for AMI. The fasting whole blood was drawn in vacutainer containing EDTA. Before analysis, samples were washed and transmethylated. We performed the quantification of different FA by gas chromatography associated with flame ionization detector (GCFID). Results: We obtained results in control healthy patients to be used as reference values. In the AMI group, levels of omega-6 were significantly higher (p 0.05) for C18:2n6 and C18:3n6 than the reference population and omega-3 values were significantly lower (p 0.01) compared to reference value for C22:6n3. The omega-3 index was lower and the ratio omega-6/omega-3 was higher in AMI group compared to reference values. Conclusions: We have established reference value for FA and have compared these values with the results obtained in AMI population. FA determination is a new tool we are able to use and to process in our laboratory which can help the clinician to screen patients with the highest cardiovascular risks because of the implication of FA in the etiopathogeny of atherosclerosis.

International audience; Background: Pioneer studies demonstrate the impact of extreme sport load on the human brain, leading to threatening conditions for athlete's health such as cerebral edema. The investigation of brain water diffusivity, allowing the measurement of the intercellular water and the assessment of cerebral edema, can give a great contribution to the investigation of the effects of extreme sports on the brain. We therefore assessed the effect of supra-physiological effort (extreme distance and elevation changes) in mountain ultra-marathons (MUMs) athletes combining for the first time brain magnetic resonance imaging (MRI) and blood parameters.Methods:This longitudinal study included 19 volunteers (44.2 ± 9.5 years) finishing a MUM (330 km, elevation + 24000 m). Quantitative measurements of brain diffusion-weighted images (DWI) were performed at 3 time-points: Before the race, upon arrival and after 48 h. Multiple blood biomarkers were simultaneously investigated. Data analyses included brain apparent diffusion coefficient (ADC) and physiological data comparisons between three time-points.Results:The whole brain ADC significantly increased from baseline to arrival (p = 0.005) and then significantly decreased at recovery (p = 0.005) to lower values than at baseline (p = 0.005). While sodium, potassium, calcium, and chloride as well as hematocrit (HCT) changed over time, the serum osmolality remained constant. Significant correlations were found between whole brain ADC changes and osmolality (p = 0.01), cholesterol (p = 0.009), c-reactive protein (p = 0.04), sodium (p = 0.01), and chloride (p = 0.002) plasma level variations.Conclusions:These results suggest the relative increase of the inter-cellular volume upon arrival, and subsequently its reduction to lower values than at baseline, indicating that even after 48 h the brain has not fully recovered to its equilibrium state. Even though serum electrolytes may only indirectly indicate modifications at the brain level due to the blood brain barrier, the results concerning osmolality suggest that body water might directly influence the change in cerebral ADC. These findings establish therefore a direct link between general brain inter-cellular water content and physiological biomarkers modifications produced by extreme sport.

Background While a moderate exercise benefits on the cardiovascular system, consequences of a supraphysiological effort are not yet clear. Objective The aim of our study was to evaluate the consequences of an ultra-marathon on cardiac and muscles markers (CK, myoglobin, hsTnT, NT-proBNP, copeptin, H-FABP) but also markers of inflammation CRP DFO, GB) and of renal function (urinary NGAL and plasma and urinary creatinine). This project also studied the evolution of new cardiac fibrosis biomarkers: ST2 and Gal-3. Design Longitudinal, cohort study. Setting The Tor des Geants (TdG), a 330 km long ultra distance trail running, with +24,000 m elevation changes in the Valley of Aosta (Italy), is considered as one of the most difficult mountain marathon race in the world. The maximum time allowed to complete the race is 150 h. Patients (or Participants) 51 participants attending the TdG have been followed and the study was conducted on 33 subjects having reached at least half of the race (148.7 km). Interventions (or Assessment of Risk Factors) Repeated blood and urine samples collection were carried out at four key time points: before, during (mid-point) and after the race (less than 1 h) and 3 days after the end of the race. Main Outcome Measurements Biological samples were collected at these four different times. Several biomarkers were assayed on different analyzers such COBAS®, KRYPTOR®, VIDAS® and ETIMAX®, meanwhile, the ST2 and Gal-3 were measured manually. Results During the TdG, the plasma levels of cardiac markers, muscle and inflammation have increased significantly to halfway. Meanwhile, the markers of renal function have only slightly varied, excepting plasma creatinine. Conclusions The study suggests that there is no permanent structural damage at the myocardium level. However, the low pace adopted by the tired runners caused an inflammatory response as well as muscle damage less important than a shorter race.

Background: The study was conducted to evaluate the analytical and clinical performance of the VIDAS (R) 25-OH Vitamin D Total assay. The clinical performance of the assay was compared with four other immunoassays against the results of two different liquid chromatography/mass spectrometry methods (LC-MS/MS)-standardized to NIST reference materials. Methods: VIDAS (R) 25-OH Vitamin D Total assay precision, linearity, detection limits and sample matrix comparison were assessed following CLSI guidelines. For method comparison, a total of 150 serum samples ranging from 7 to 92 ng/mL were analyzed by all the methods. Correlation was studied using Passing-Bablok regression and Bland-Altman analysis. The concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and the reference LC-MS/MS method. In addition, samples containing endogenous 25(OH) D 2 were used to assess each immunoassay's ability to detect this analyte. Pregnancy and hemodialysis samples were used to the study the effect of vitamin D binding protein (DBP) concentration over VIDAS (R) assay performance. Results: The VIDAS (R) 25-OH Vitamin D Total assay showed excellent correlation to the LC-MS/MS results (y = 1.01x+0.22 ng/mL, r=0.93), as obtained from two different sites and distinct LC-MS/MS methods. The limit of quantification was determined at 8.1 ng/mL. Cross-reactivity for 25(OH) D 2 was over 80%. At concentrations of 10.5, 26 and 65.1 ng/mL, within-run CVs were 7.9%, 3.6% and 1.7%, while total CVs (between runs, calibrations, lots and instruments) were 16.0%, 4.5% and 2.8%. The VIDAS (R) performance was not influenced by altered DBP levels, though under-recovery of 25(OH) D as compared to LC-MS/MS was observed for hemodialysis samples. Conclusions: The VIDAS (R) 25-OH Vitamin D Total assay is therefore considered suitable for assessment of vitamin D status in clinical routine.

Background Accurate quantitation of aldosterone is essential for screening, diagnosis and subtype classification in primary aldosteronism. A simple, sensitive method for aldosterone in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated. Methods Plasma samples were diluted with water containing d 7 -aldosterone as internal standard. The samples were extracted with methyl-tert-butyl-ether (MTBE) on SLE cartridges. Separation was carried out on a Luna C18 (2) column using a methanol–water gradient. Detection was performed in the negative electrospray multiple reaction monitoring (MRM) quantitation. The use of water-based calibrators was evaluated against calibrators prepared in steroid-free serum. Results The assay was linear up to 3265 pmol/L with an LOQ of approximately 40 pmol/L. Within-run and between-run precision for plasma aldosterone were less than 10% except at low level near LOQ but were still less than 14.7% (Westgard's desirable specification). The mean recovery of the analyte added to plasma was greater than 97.7% and matrix effects were less than 4%. Comparison with another LC-MS/MS method was performed on a more sensitive instrument (ABSciex TQ 5500) and gave the equation API 3000 = 0.957 × TQ 5500 + 12.6, linear regression r 2 = 0.974 ( n = 43). An estimation of the reference interval for adults was established on a group of healthy volunteers ( n = 53). Calibration with water-based calibrators was validated and can be used for measurement of aldosterone by LC-MS/MS. Conclusions This method is reliable, easy to perform on plasma specimens in a clinical environment and is attractive because of its simplicity.

Summary Introduction Cardiopathies are the world's leading cause of mortality and morbidity. Although rare, cardiovascular accidents can occur during intense and infrequent sporting activity, particularly among those who are unaware of their heart condition. The development of cardiospecific biochemical markers has led to a reconsideration of the role of biology in the diagnosis of cardiovascular illnesses. The aim of this study therefore was, through the use of cardiac biomarker assays, to highlight the impact of sustained physical effort in the form of intense and prolonged concentric isokinetic exercise and to research potential cardiovascular risks. Materials and methods Eighteen subjects participated in a maximal concentric isokinetic exercise involving 30 knee flexion–extensions for each leg. Five blood tests were taken to study the kinetics of the cardiac biomarkers. Haemodynamic parameters were measured continuously using a Portapres, and respiratory parameters were measured using a Sensormedics Vmax 29C. Results The results showed significant increases in the creatine kinase, myoglobin, homocysteine and haemoglobin cardiac markers. Evolutionary trends were also observed for the following biomarkers: NT-proBNP, myeloperoxydase and C-reactive protein. All the physiological parameters measured presented statistically significant changes. Conclusion Isokinetic effort leads to the release of cardiac markers in the blood, but these do not exceed the reference values in healthy subjects. Maximal concentric isokinetic exercise does not, therefore, lead to an increased risk of cardiovascular pathologies.

Background: The study was conducted to evaluate the technical and clinical performance of the VITROS® Immunodiagnostic Products 25-OH Vitamin D Total Assay, and compare it with the performance of five marketed automated assays and a liquid chromatography/mass spectrometry reference method (LC-MS/MS). Methods: Three hundred patient serum samples were used to compare the correlation of the VITROS® 25-OH Vitamin D Total Assay with both the other immunoassays and the LC-MS/MS method, using Passing-Bablok regression and Bland-Altman analyses. Concordance of the diagnosis of vitamin D status was calculated to test the agreement between the different assays. In addition, samples containing vitamin D2 were used to test the assay’s ability to detect the D2 form of the vitamin. Results and conclusions: These results from the VITROS® 25-OH Vitamin D Total Assay generally correlated well with those from most of the marketed immunoassays. Cross-reactivity of the D2 form was calculated as being close to 100%. Additionally, we found substantial variability in performance amongst the various assays, which suggests the need for optimisation and recalibration of commercial methods.

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.

Introduction The tendon is a tissue which does not heal easily. Recently, several studies have demonstrated the positive effects of platelets on the healing process of tendons. A local injection of platelet-rich plasma (PRP), which releases in situ many growth factors, has the potentiality to enhance the tendon healing process. The aim of our experiment was to ascertain by an original mechanical measure whether the use of PRP was of interest for accelerating the healing process of rats9 Achilles tendons after surgical induced lesion. Methods A 5 mm defect was surgically induced in 90 rats9 Achilles tendon. Rats were divided into two groups of 45: (A) control (no treatment) and (B) PRP treatment. Rats of group B received a PRP injection in situ after the surgery. Afterwards, rats of both groups were placed in their cages without immobilisation. After 5, 15 and 30 days, 10 traumatised Achilles tendons of each group were dissected and removed. Immediately after sampling, tendons were submitted to a biomechanical tensile test up to rupture, using a ‘Cryo-jaw’. After that, transcriptomic analyses were made on the tendon samples, to study the expression of type III collagen, matrix metalloproteases and tenomodulin. A hydroxyproline dosage was done to quantify the collagen in the tendon during its healing process. Tendons of the 15 remaining rats of each group were subjected to a histological study, respectively at day 5, 15 and 30 (five rats for each time). Results We demonstrated that the force necessary to induce tendon rupture during biomechanical tensile test study was greater for tendons which had been submitted to an injection of PRP compared to the control group: +19% (day 5), +30% (day 15) and +43% (day 30). Histological study showed that PRP could enhance cells proliferation, angiogenesis and collagen organisation. Our biochemical analyses did not explain beneficial effects of PRP. Indeed, there was no significant difference neither between the expression of different studied genes, nor in the quantity of hydroxyproline between both groups. Conclusion This experimentation has shown that a PRP injection could accelerate the tendons healing process and improve its quality.

Tendon lesions are among the most frequent musculoskeletal pathologies. Vascular endothelial growth factor (VEGF) is known to regulate angiogenesis. VEGF-111, a biologically active and proteolysis-resistant splice variant of this family, was recently identified. This study aimed at evaluating whether VEGF-111 could have a therapeutic interest in tendon pathologies. Surgical section of one Achilles tendon of rats was performed before a local injection of either saline or VEGF-111. After 5, 15 and 30 days, the Achilles tendons of 10 rats of both groups were sampled and submitted to a biomechanical tensile test. The force necessary to induce tendon rupture was greater for tendons of the VEGF-111 group (p