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.

Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen.

Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.

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.

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.

Analytical performance specifications for the measurement uncertainty of 24,25-dihydroxyvitamin D examinations.

OBJECTIVES: The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D's BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated. METHODS: Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K3-EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point. RESULTS: Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at p<0.05 would need a relative measurement uncertainty (u%)<14.9% while at p<0.01, relative measurement uncertainty should be <10.5%. CONCLUSIONS: We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods.

Parathormone, bone alkaline phosphatase and 25-hydroxyvitamin D status in a large cohort of 1200 children and teenagers.

Objectives: 25-Hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH) and bone alkaline phosphatase (BALP) are biomarkers of calcium/phosphate metabolism and bone turnover. Although vitamin D deficiency is a well-known cause of secondary hyperparathyroidism, few studies have considered vitamin D status when establishing reference ranges. In this study, we report PTH levels according to the vitamin D status and BALP levels in a large cohort of 1200 children. Additionally, we provide PTH pediatric reference values according to 25(OH)D status as well as BALP pediatric reference ranges.Methods: Serum samples from 1200 children (equally distributed from 5 months to 20 years old) who underwent blood sampling for allergy exploration were used to quantify 25(OH)D, PTH and BALP.Results: The percentage of vitamin D deficient children (<20 ng/ml) progressively increased during childhood starting from 7% in the 0 to 2 year-old subgroup to a mean of at least 50% among teenagers. PTH levels inversely mirrored 25(OH)D concentrations for all age and gender subgroups, and 25(OH)D deficient subgroups presented higher PTH levels than their non-deficient counterparts. In the non-deficient 25(OH)D population, PTH levels were the highest at 11 years old for girls and 14 years old for boys. BALP results were slightly increased during childhood and showed a constant decrease during teenage years starting from 12 years old for girls and 14 years old for boys.Conclusion: Our results highlight the inverse relationship between PTH and 25(OH)D in children and the need for a well characterized 25(OH)D population to establish pediatric reference ranges for PTH.

Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects.

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 <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >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.

A fast and simple method for simultaneous measurements of 25(OH)D, 24,25(OH)2D and the Vitamin D Metabolite Ratio (VMR) in serum samples by LC-MS/MS.

BACKGROUND: Rapid, easy and reliable measurement of the major vitamin D metabolites is required in order to fulfill the needs of a clinical routine laboratory. To overcome these challenges, we have developed and validated a LC-MS/MS method for the quantification of 25-hydroxyvitamin D2 and D3, epi-25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. METHODS: Sample preparation was based on precipitation and centrifugation of 100µL of patient serum, followed by injection into the LC-MS/MS system. Samples from Vitamin D Standardization Program (n=80) and patient samples (n=281) have been compared with a reference LC-MS/MS method. For the analytical validation NIST and Labquality quality control materials were used. RESULTS: Mean intra-assay and inter-assay imprecision were <6.0 and 6.4% and mean recoveries were within 95-104%. LOQ's were 0.5µg/L for 24,25(OH)2D3, 1.1µg/L for 25(OH)D3 and epi-25(OH)D3 and 1.7µg/L for 25(OH)D2. A 3% bias obtained between the proposed and the reference method satisfies Vitamin D Standardization Program recommendations. CONCLUSIONS: We present a rapid, easy, reliable and cost-effective method completely adequate for routine testing, which permits the measurement of the ratio of 24,25-dihydroxyvitamin D to 25-hydroxyvitamin D, Vitamin D Metabolite Ratio (VMR), in serum samples.

Carbon, nitrogen and sulphur isotopic fractionation in captive juvenile hooded seal (Cystophora cristata): Application for diet analysis.

RATIONALE: Intrinsic biogeochemical markers, such as stable isotope ratios of carbon, nitrogen and sulphur, are increasingly used to trace the trophic ecology of marine top predators. However, insufficient knowledge of fractionation processes in tissues continues to hamper the use of these markers. METHODS: We performed a controlled feeding experiment with eight juvenile hooded seals (Cystophora cristata) that were held on a herring-based diet (Clupea harengus) for two years. Stable isotope ratios were measured via isotope ratio mass spectrometry in three of their tissues and related to values of these markers in their diet. RESULTS: Diet-tissue isotope enrichment (trophic enrichment factor, TEF) values between dietary herring and seal tissues for carbon (Δ13 C) were +0.7 ‰ for red blood cells, +1.9 ‰ for hair and +1.1 ‰ for muscle. The TEFs for nitrogen trophic (Δ15 N) were +3.3 ‰ for red blood cells, +3.6 ‰ for hair and +4.3 ‰ for muscle. For sulphur, the Δ34 S values were +1.1 ‰ for red blood cells, +1.0 ‰ for hair and +0.9 ‰ for muscle. CONCLUSIONS: These enrichment values were greater than those previously measured in adult seals. This increase may be related to the higher rate of protein synthesis and catabolism in growing animals. This study is the first report on sulphur isotope enrichment values for a marine mammal species.