Development and validation of an LC-MS/MS method for the simultaneous quantitation of angiotensin (1-7), (1-8), (1-9) and (1-10) in human plasma.

Cardiovascular diseases have cast a significant negative impact on the lives of millions worldwide. Over the years, extensive efforts have been dedicated to enhancing diagnostic and prognostic tools for these diseases. A growing body of evidence indicates that the angiotensin convertase enzyme (ACE) and the angiotensin convertase enzyme 2 (ACE2), and angiotensin peptide levels could hold a pivotal role in assisting clinicians with the management of cardiovascular conditions, notably hypertension and heart failure. However, despite the considerable body of knowledge in this domain, a void remains in the field of analytical methodologies for these molecules. In this study, we present a fully validated LC-MS/MS method for the precise quantitation of plasma angiotensin (1-7), (1-8), (1-9), and (1-10), following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI). Our method not only enables the accurate quantification of angiotensin peptides but also provides a means to assess ACE and ACE2 activity. Remarkably, our method achieved a Lower Limit of Measurement Interval (LLMI) as low as 5 pg/mL. This has enabled the detection of angiotensin (1-7), (1-8), (1-9) and (1-10) and the accurate quantitation of angiotensin (1-7), (1-8) and (1-10) in all analyzed groups, including healthy controls, patients with high blood pressure, and patients with chronic kidney disease. To our knowledge, our method represents the most sensitive approach allowing for simultaneous quantitation of these four angiotensin peptides. A distinct advantage of our method, when compared to immunoassays, is its high sensitivity combined with comprehensive chromatographic separation of all currently known angiotensin peptides. This combination translates to an exceptional level of selectivity, underscoring the value and potential of our methodology in advancing cardiovascular disease research.

Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva.

A growing body of literature describes the potential effects of circadian disruption on human health. Indeed, psychiatric diseases, metabolic syndrome, and cancers may be linked to disturbance of the circadian rhythm. Currently, the best practice to assess circadian rhythm is the measurement of melatonin levels. Our goal was thus to develop and validate a highly sensitive LC-MS/MS method to follow salivary melatonin levels throughout the day and night. Our method reached a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. To our knowledge, it is the most sensitive method allowing quantitation of melatonin in saliva. Saliva, obtained from passive drooling or salivette, was extracted by an efficient and quick liquid-liquid extraction with no further cleanup needed. The method was validated according to the European Medicines Agency (EMA) guidelines and provided excellent results regarding accuracy, precision, linearity, selectivity, and specificity. Comparison between radioimmunoassay and our method was performed and showed differences at low levels, most likely due to cross-reactivity with other indols. To assess daytime melatonin levels in humans, salivary melatonin levels of ten volunteers were monitored throughout the day and showed lower daytime levels than reported in previous studies.

Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications.

Immunocapture is now a well-established method for sample preparation prior to quantitation of peptides and proteins in complex matrices. This short review will give an overview of some clinical applications of immunocapture methods, as well as protocols with and without enzymatic digestion in a clinical context. The advantages and limitations of both approaches are discussed in detail. Challenges related to the choice of mass spectrometer are also discussed. Top-down, middle-down, and bottom-up approaches are discussed. Even though immunocapture has its limitations, its main advantage is that it provides an additional dimension of separation and/or isolation when working with peptides and proteins. Overall, this short review demonstrates the potential of such techniques in the field of proteomics-based clinical medicine and paves the way for better personalized medicine.

Evolution of 17-ß-estradiol, estrone and estrone-sulfate concentrations in late pregnancy of different breeds of mares using Liquid Chromatography and Mass Spectrometry.

This study describes 17-ß-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's parity and age on estrogens concentrations were investigated. Peak of E2 was observed at 5 months (median: 46.4 pg/mL; maximum: 201.5 pg/mL). A strong correlation was observed between E1 and E1S (p < 0.0001, r = 0.85). Peak of E1 (median: 571.0 pg/mL; maximum: 1641.9 pg/mL) and E1S (median: 573.6 ng/mL; maximum: 997.6 ng/mL) concentrations was observed at the 5th month and then E1S decreased quicker than E1 until the end of pregnancy. Higher E2 and E1 concentrations were observed in SJ than in SPB mares between the 6th and the 8th months. No difference between breeds was observed for E1S monthly evolution. Estrogen peak values were all observed at 5 months. Unlike recent LC-MS studies, E1S values observed here were in the same range than those previously established using immuno-assays. After the 6th month, E1S decreased quicker than E1. Effect of breed only observed on non-sulfonated estrogens should be further confirmed. These findings confirm that sulfonation activity of the allantochorion may be limited after the 6th month.

Kinetics of Cardiac Remodeling and Fibrosis Biomarkers During an Extreme Mountain Ultramarathon.

Objectives: The effects of ultra-distance on cardiac remodeling and fibrosis are unclear. Moreover, there are no data reporting the kinetics of cardiac alterations throughout the event and during recovery. Our aim was to investigate the kinetics of biological markers including new cardiac fibrosis biomarkers suppression of tumorigenicity 2 (ST2) and galectin-3 (Gal-3) during and after an extreme mountain ultramarathon. Methods: Fifty experienced runners participating in one of the most challenging mountain ultramarathons (330 km, D+ 25,000 m) were enrolled in our study. Blood samples were collected at four time points: before (Pre-), at 148 km (Mid-), at the finish line (Post-), and 3 days after the recovery period (Recov-). Results: The cardiac fibrosis biomarkers (ST2 and Gal-3) increased from Pre- to Mid-. During the second half, ST2 remained higher than pre-values as opposed to Gal-3. Necrosis, ischemia, and myocyte injury biomarkers increased until Mid- then decreased but remained higher at Recov- than Pre-values. Oxidative stress appeared at Mid-. Lipid peroxides remained higher at Recov- compared to Pre-. The maximal value in most of these biomarkers was observed at Mid- and not at Post-. Conclusions: The present study supports biphasic kinetics of cardiac fibrosis biomarkers, with a relative recovery during the second half of the event that seems specific to this extreme event. Overall, performing at such an extreme ultramarathon seems less deleterious for the heart than shorter events.

Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons.

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 deviation <15% and relative bias <15%, 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.

Evolution of the slopes of ST2 and galectin-3 during marathon and ultratrail running compared to a control group.

Background Previous studies have suggested that exercising may induce cardiac damage. Galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (ST2) are very interesting biomarkers for heart failure and myocardial fibrosis. We aimed to compare the kinetics of emerging fibrosis cardiac biomarkers as Gal-3 and ST-2 in endurance runners, and recreational runners before and after a running event represented by a marathon and an ultratrail event. Methods Blood samples were taken from 19 healthy non-elite marathon runners (42 km), 27 ultratour runners (67 km), and 14 recreational runners who represented the control group (10 km) just before the run (T0), just after (T1) and 3 h after (T2), in order to analyze Gal-3, ST2, hsTnT, NT-proBNP, CKMB and hsCRP. We compared the percentage of evolution and the slopes obtained from T0 to T1 (pT0T1) and from T1 to T2 (pT1T2), between the different groups of runners participating in three different races. Results Plasma cardiac biomarker concentrations increased significantly from baseline to immediately post-exercise and most of the time decreased over the subsequent 3-h period. For pT0T1 and pT1T2, the markers Gal-3 and ST2 showed a significant difference between types of run (p < 0.05 and p < 0.0001, respectively). During the recovery time, Gal-3 returned to the baseline values but not ST2 which continued to increase. Conclusions Gal-3 and ST2 are considered as a reflection of cardiac fibrosis and remodeling. The evolution of both was different, particularly after the recovery time. ST2 values exceeding cutoff values at any time.

Sunscreens block cutaneous vitamin D production with only a minimal effect on circulating 25-hydroxyvitamin D.

A 50+ SPF sunscreen decreased significantly cutaneous vitamin D production following a single narrow-band (nb)UVB exposure, independently from the body surface area exposed. In contrast, the circulating 25(OH)D3 levels were only minimally affected. It is probable that another endogenous source of precursors is selected when skin-originated precursors are lacking. PURPOSE: Sunscreen use, highly advocated for preventing cutaneous carcinogenesis, is potentially leading to an aggravation of vitamin D deficiency with its consequences on bone health. The effect of sunscreens on circulating vitamin D levels remains debated. This study investigated the effect of sunscreen on cutaneous vitamin D production and circulating 25(OH)D3 levels, according to different body surface areas (BSA). METHODS: Vitamin D and 25(OH)D3 levels were measured in four groups exposed to a single nbUVB exposure on 9% (group I: head and hands), 23% (group II: head, hands and arms), 50% (group III: head, hands, arms and legs) and 96% (group IV: total body) of the body surface without and with a 50+ sun protection factor sunscreen. RESULTS: Sunscreen use decreased by 83, 88.3, 75.7 and 92.5% the cutaneous vitamin D production in groups I to IV, respectively, but only by 13.2, 10.5, 7.7 and 10.4% the values of circulating 25(OH)D3, correspondingly. CONCLUSIONS: Although a 50+ sunscreen decreases significantly cutaneous vitamin D production following a single nbUVB exposure, and independently from the BSA, the circulating 25(OH)D3 levels were only minimally affected. This could be explained by a switch to another endogenous source of precursors. Short-term sunscreen use probably does not affect circulating vitamin D levels and hence does not increase the risk for osteoporosis. The effect of long-term sunscreen use remains however to be determined.

New biomarkers for primary mitral regurgitation.

BACKGROUND: Mitral regurgitation is a frequent valvular heart disease affecting around 2.5 % of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered as a passive ongoing pathophysiological process and little is known about its physiological deregulation. The purpose of this study was to highlight new biomarkers of mitral regurgitation in order to decipher the underlying pathological mechanism as well as to allow the diagnosis and the monitoring of the disease. RESULTS: Modulation of various blood proteins expression was examined in patients suffering from different grades of mitral regurgitation (mild, moderate and severe) compared to healthy controls. To this end, several routine clinical assays and the multi analyte profile technology targeting 184 proteins were used. High-density lipoprotein, apolipoprotein-A1, haptoglobin and haptoglobin-α2 chain levels significantly decreased proportionally to the degree of mitral regurgitation when compared to controls. High-density lipoprotein and apolipoprotein-A1 levels were associated with effective regurgitant orifice area and regurgitant volume. Apolipoprotein-A1 was an independent predictor of severe mitral regurgitation. Moreover, with ordinal logistic regression, apolipoprotein-A1 remained the only independent factor associated with mitral regurgitation. In addition, myxomatous mitral valves were studied by immunocytochemistry. We observed an increase of LC3, the marker of autophagy, in myxomatous mitral valves compared with healthy mitral valves. CONCLUSION: These potential biomarkers of mitral regurgitation highlighted different cellular processes that could be modified in myxomatous degenerescence: reverse cholesterol transport, antioxidant properties and autophagy.

Apolipoprotein-A1 as a damage-associated molecular patterns protein in osteoarthritis: ex vivo and in vitro pro-inflammatory properties.

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.

Critical care and vitamin D status assessment: what about immunoassays and calculated free 25OH-D?

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 bioMérieux 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.

Effects of platelet-rich plasma (PRP) on the healing of Achilles tendons of rats.

Platelet-rich plasma (PRP) contains growth factors involved in the tissular healing process. The aim of the study was to determine if an injection of PRP could improve the healing of sectioned Achilles tendons of rats. After surgery, rats received an injection of PRP (n = 60) or a physiological solution (n = 60) in situ. After 5, 15, and 30 days, 20 rats of both groups were euthanized and 15 collected tendons were submitted to a biomechanical test using cryo-jaws before performing transcriptomic analyses. Histological and biochemical analyses were performed on the five remaining tendons in each group. Tendons in the PRP group were more resistant to rupture at 15 and 30 days. The mechanical stress was significantly increased in tendons of the PRP group at day 30. Histological analysis showed a precocious deposition of fibrillar collagen at day 5 confirmed by a biochemical measurement. The expression of tenomodulin was significantly higher at day 5. The messenger RNA levels of type III collagen, matrix metalloproteinases 2, 3, and 9, were similar in the two groups at all time points, whereas type I collagen was significantly increased at day 30 in the PRP group. In conclusion, an injection of PRP in sectioned rat Achilles tendon influences the early phase of tendon healing and results in an ultimately stronger mechanical resistance.