Determination of vitamin D3 conjugated metabolites: a complementary view on hydroxylated metabolites.

Experts typically define vitamin D deficiency levels by the determination of a circulating 25-hydroxyvitamin D3-calcifediol prohormone. A large part of the population is characterized by deficient vitamin D levels (calcifediol < 20 ng mL-1) despite individuals not being affected by any disorder. Cholecalciferol (vitamin D3) and/or calcifediol supplementation is a common practice for vitamin D-deficient individuals as recommended by international scientific societies and official agencies. In the last few years, several studies have reported the presence of conjugated vitamin D3 metabolites, mainly glucuronidation and sulfation derivatives, although simultaneous quantitative measurements involving phase I and II vitamin D metabolites have not been carried out. A quantitative method based on tandem mass spectrometry detection is proposed here for the combined determination of phase I and phase II vitamin D3 metabolites in human serum. As phase I and phase II metabolites are preferentially ionized in different modes, a switching polarity mode was adopted to determine both groups of compounds in serum at high sensitivity levels (pg mL-1). The validation of this proposal was successfully accomplished by following the Center for Drug Evaluation and Research (CDER) guidelines. Its applicability was tested in a cohort of volunteers with mostly deficient baseline levels. Considering the sulfated form of calcifediol, the sum of its concentrations showed sufficient baseline vitamin D levels in all individuals, suggesting that this could be a novel strategy for vitamin D deficiency definition. Therefore, phase II metabolites are proposed to be included when evaluating the vitamin D status since they provide more information about the overall status of the vitamin D endocrine system. Nevertheless, further studies are required to confirm the biological activity of these conjugated metabolites and the suitability of this strategy for the description of vitamin D deficiency.

Comparative Study of the Efficacy of EHO-85, a Hydrogel Containing Olive Tree (Olea europaea) Leaf Extract, in Skin Wound Healing.

Olive tree (Olea europaea) leaf extract (OELE) has important antioxidant and anti-inflammatory properties, supporting its use in human clinical practice. We recently designed an amorphous hydrogel called EHO-85 (EHO indicates olive leaf extract in Spanish) containing OELE for skin ulcer treatments. Yet, its effectiveness has not been previously compared with other products used in routine clinical practice. This is necessary to evaluate its potential translation to the human clinic. Thus, in this study, the effect of EHO-85 on healing was evaluated in comparison with treatments containing Indian/Asiatic pennywort (Centella asiatica), hyaluronic acid, or dexpanthenol in a rat model. The speed of wound closure and histological parameters after seven and 14 days were analyzed. All treatments accelerated wound closure, but there were differences between them. Dexpanthenol after seven days produced the highest epithelialization and the lowest inflammation and vascularization. EHO-85 also promoted epithelialization and reduced vascularization. After 14 days, wounds treated with EHO-85 showed less inflammation and higher levels of collagen in the extracellular matrix. This indicates a higher degree of maturity in the regenerated tissue. In conclusion, the effect of EHO-85 on healing was equal to or superior to that of other treatments routinely used in human clinical practice. Therefore, these results, together with previous data on the effects of this hydrogel on ulcer healing in humans, indicate that EHO-85 is a suitable, low-cost, and efficient therapeutic option for wound healing.

Proof of Concept on Functionality Improvement of Mesenchymal Stem-Cells, in Postmenopausal Osteoporotic Women Treated with Teriparatide (PTH1-34), After Suffering Atypical Fractures.

Osteoporosis long-term treatment with nitrogen-containing bisphosphonates, has been associated with uncommon adverse effects, as atypical femoral fractures (AFF). Thus, treatment with teriparatide (TPTD; fragment of human parathyroid hormone; PTH1-34) has been proposed for such patients. Besides its anabolizing effect on bone, TPTD may affect stem-cell mobilization and expansion. Bone marrow mononuclear cells (BMMNC) were isolated from five women that had suffered AFF associated to bisphosphonate treatment, before and after 6 months of TPTD therapy. The presence of mesenchymal stromal cells (CD73, CD90 and CD105 positive cells), gene expression of NANOG, SOX2 and OCT4, proliferation, senescence and capacity to differentiate into osteoblasts and adipocytes were analyzed. After TPTD treatment, BMMNC positive cells for CD73, CD90 and CD105 increased from 6.5 to 37.5% (p < 0.05); NANOG, SOX2 and OCT4 were upregulated, being statistically significant for NANOG (p < 0.05), and cells increased proliferative capacity more than 50% at day 7 (p < 0.05). Senescence was reduced 2.5-fold (p < 0.05), increasing differentiation capacity into osteoblasts and adipocytes, with more than twice mineralization capacity of extracellular matrix or fat-droplet formation (p < 0.05), respectively. Results show that TPTD treatment caused BMMNC "rejuvenation", increasing the number of cells in a more undifferentiated stage, with higher differentiation potency. This effect may favor TPTD anabolic action on bone in such patients with AFF, increasing osteoblast precursor cells. Such response could also arise in other osteoporotic patients treated with TPTD, without previous AFF. Furthermore, our data suggest that TPTD effect on stromal cells may have clinical implications for bone-regenerative medicine. Further studies may deepen on this potential.

High Serum Retinol as a Relevant Contributor to Low Bone Mineral Density in Postmenopausal Osteoporotic Women.

There is controversial information about the impact of vitamin A on bone. Some epidemiological studies show that excessive intake of vitamin A, or an excess of serum vitamin A, has related with adverse impact on bone mass; however, other studies did not find these links, and some authors have proposed that this vitamin might promote a better bone health. The present work aims to contribute to clarify the real role of vitamin A in bone tissue. For this purpose, a cross-sectional study of 154 osteoporotic non-treated postmenopausal women (> 65 years old) was carried out. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. We assessed concentrations of serum retinol, osteocalcin, parathyroid hormone, alkaline phosphatase, calcium, and phosphorus. We also studied demographic and anthropometric parameters. Spearman's correlations between retinol levels and other variables found negative correlations with BMD in both lumbar spine (R = - 0.162, P < 0.01) and femoral neck (R = - 0.182, P < 0.01), as well as alkaline phosphatase (R = - 0.110; P < 0.05) and phosphorus (R = - 0.110; P < 0.05). A positive correlation between retinol and fertile window was observed (R = 0.158; P < 0.01). After multivariable adjustment, we still found a negative correlation between serum retinol and BMD, both at the lumbar spine (R = - 0.210; P < 0.01) and at the femoral neck (R = - 0.324, P < 0.001). It is concluded that elevated serum-retinol levels are associated with an increased risk of low bone mass and thus with osteoporotic fractures. Therefore, osteoporosis-risk assessment should include quantification of serum metabolite of vitamin A.

Transcriptomic Analyses of Adipocyte Differentiation From Human Mesenchymal Stromal-Cells (MSC).

Adipogenesis is a physiological process required for fat-tissue development, mainly involved in regulating the organism energetic-state. Abnormal distribution-changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal-cells (MSC) induced and not induced to differentiate into adipocytes by next (second)-generation sequencing. A total of 2,866 differentially expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane-organelle biosynthesis, and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation, and differentiation. Analyses of mRNA-miRNA interaction showed that repressed miRNA-encoding genes can act downregulating PPARG-related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA-encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose-tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type-2 diabetes and osteoporosis. J. Cell. Physiol. 232: 771-784, 2017. © 2016 Wiley Periodicals, Inc.

Calcifediol or Corticosteroids in the Treatment of COVID-19: An Observational Study.

Medical treatment of coronavirus 19 disease (COVID-19) is a therapeutic challenge. The available data strongly suggest that calcifediol treatment may reduce the severity of COVID-19, and corticosteroids are the treatment of choice worldwide for severe COVID-19. Both have a very similar action profile, and their combined use in patients may modify the contribution of each administered compound. OBJECTIVE: To evaluate how treatment with calcifediol and/or corticosteroids in medical practice modified the need for ICU admission, death, or poor prognosis of patients hospitalized with COVID-19 during the first outbreaks. DESIGN, PATIENTS AND SETTING: A retrospective observational cohort study of patients admitted for COVID-19 to the Pneumology Unit of the Hospital Universitario Reina Sofía (Córdoba, Spain). INTERVENTIONS: Patients were treated with calcifediol or/and corticosteroids with the best available therapy and standard care, according to clinical practice guidelines. MEASUREMENTS: Admission to the intensive care unit (ICU) or death during hospitalization and poor prognosis. RESULTS: Seven hundred and twenty-eight patients were included. According to the treatment received, they were included in four groups: calcifediol (n = 68), glucocorticoids (n = 112), both (n = 510), or neither (n = 38). Of the 578 patients treated with calcifediol, 88 were admitted to the ICU (15%), while of the 150 not treated with calcifediol, 39 required ICU admission (26%) (p < 0.01). Among the patients taking calcifediol without glucocorticoids, only 4 of 68 (5.8%) required ICU admission, compared to 84 of 510 (16.5%) treated with both (p = 0.022). Of the 595 patients who had a good prognosis, 568 (82.01%) had received treatment with calcifediol versus the 133 patients with a poor prognosis, of whom 90 (67.66%) had received calcifediol (p < 0.001). This difference was not found for corticosteroids. INTERPRETATION: The treatment of choice for hospitalized patients with moderate or mild COVID-19 could be calcifediol, not administering corticosteroids, until the natural history of the disease reaches a stage of hyperinflammation.

Calcifediol Cornerstone of the Vitamin D Endocrine System.

It is likely that rickets has afflicted humanity since the dawn of time, but it was first described in great detail in the mid-17th century [...].

Comparison of calcifediol with vitamin D for prevention or cure of vitamin D deficiency.

Vitamin D deficiency remains prevalent, with about 7% of the world's population living with severe vitamin D deficiency and about one third with mild deficiency. We compare the relative merits of calcifediol or 25-hydroxyvitamin D (25OHD) compared to vitamin D itself for supplementation as to prevent or cure vitamin D deficiency. The intestinal absorption of calcifediol is nearly 100% and thus higher than that of vitamin D itself. Moreover, calcifediol is absorbed by the intestinal cells and transported through the portal vein and thus immediately accessible to the circulation, while vitamin D is transported with chylomicrons through the lymph system. Therefore, in case of fat malabsorption or after bariatric surgery, calcifediol is much better absorbed in comparison with vitamin D itself. Serum 25OHD increases linearly with increasing doses of calcifediol, whereas serum 25OHD reaches a plateau when higher oral doses of vitamin D are used. Calcifediol, on a weight basis, is about 3 times more potent than vitamin D in subjects with mild vitamin D deficiency. This potency is even 6-8 times higher than vitamin D when baseline serum 25OHD is higher or when large doses are compared. In conclusion, calcifediol is an alternative option to correct vitamin D deficiency and may even be the preferred strategy in case of intestinal fat malabsorption, after bariatric surgery or in case of other conditions with suspected impaired 25-hydroxylase activity in the liver.

Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

DPP4 may play a relevant role in MSC differentiation into osteoblasts or adipocytes. Dipeptidyl peptidase 4 (DPP4) inhibitors (DPP4i), such as sitagliptin and vildagliptin, are used as antidiabetic drugs. However, vildagliptin is not a specific DPP4i and also inhibits DPP8/9, which is involved in energy metabolism and immune regulation. The aim of this study is to evaluate how sitagliptin, vildagliptin or 1G244 (a DPP8/9 specific inhibitor) may influence cell viability, as well as osteogenic and adipogenic differentiation in human mesenchymal stem cells (MSC). Viability, apoptosis, osteoblastogenesis and adipogenesis markers, as well as protein synthesis of ß-catenin, were studied in MSC cultures induced to differentiate into osteoblasts or adipocytes in the presence or absence of sitagliptin, vildagliptin or 1G244. The two tested DPP4i did not affect MSC viability, but 1G244 significantly decreased it in MSC and osteoblast-induced cells. Additionally, 1G244 and vildagliptin inhibited osteogenesis and adipogenesis, unlike sitagliptin. Therefore, inhibition of DPP4 did not affect MSC viability and differentiation, whereas inhibition of DPP8/9 negatively affected MSC. To the best of our knowledge, these results show for the first time that DPP8/9 have an important role in the viability and differentiation of human MSC. This data can be considered for human clinical use of drugs affecting DPP8/9 activity.

EHO-85, Novel Amorphous Antioxidant Hydrogel, Containing Olea europaea Leaf Extract-Rheological Properties, and Superiority over a Standard Hydrogel in Accelerating Early Wound Healing: A Randomized Controlled Trial.

Many advanced wound healing dressings exist, but there is little high-quality evidence to support them. To determine the performance of a novel amorphous hydrogel (EHO-85) in relation to its application, we compared its rheological properties with those of other standard hydrogels (SH), and we assessed the induction of acceleration of the early stages of wound healing as a secondary objective of a prospective, multicenter, randomized, observer-blinded, controlled trial. The patients were recruited if they had pressure, venous, or diabetic foot ulcers and were treated with EHO-85 (n = 103) or VariHesive® (SH) (n = 92), and their response was assessed by intention-to-treat as wound area reduction (WAR (%)) and healing rate (HR mm2/day) in the second and fourth weeks of treatment. Results: EHO-85 had the highest shear thinning and G'/G″ ratio, the lowest viscous modulus, G″, and relatively low cohesive energy; EHO-85 had a significantly superior effect over SH in WAR and HR, accelerating wound healing in the second and fourth weeks of application (p: 0.002). This superiority is likely based on its optimal moisturizing capacity and excellent pH-lowering and antioxidant properties. In addition, the distinct shear thinning of EHO-85 facilitates spreading by gentle hand pressure, making it easier to apply to wounds. These rheological properties contribute to its improved performance.

Hard-to-Heal Wound Healing: Superiority of Hydrogel EHO-85 (Containing Olea europaea Leaf Extract) vs. a Standard Hydrogel. A Randomized Controlled Trial.

Chronic wounds, especially those that are hard-to-heal, constitute a serious public-health problem. Although progress has been made in the development of wound dressings for healing, there is little high-quality evidence of their efficacy, with no evidence of superiority in the use of one hydrogel over another. To evaluate the superiority of a hydrogel (EHO-85), containing Olea europaea leaf extract (OELE), over a standard hydrogel (SH), the promotion and/or improvement of healing of difficult-to-heal wounds was compared in a prospective, parallel-group multicenter, randomized, observer-blinded, controlled trial ("MACAON"). Non-hospitalized patients with pressure, venous or diabetic foot-ulcers difficult-to-heal were recruited and treated with standard care, and EHO-85 (n = 35) or VariHesive (n = 34) as SH. Wound-area reduction (WAR; percentage) and healing rate (HR; mm2/day) were measured. EHO-85 showed a statistically significant superior effect over VariHesive. At the end of the follow-up period, the relative WAR decreased by 51.6% vs. 18.9% (p < 0.001), with a HR mean of 10.5 ± 5.7 vs. 1.0 ± 7.5 mm2/day (p = 0.036). EHO-85 superiority is probably based on its optimal ability to balance the ulcer bed, by modulating pH and oxidative stress. That complements the wetting and barrier functions, characteristics of conventional hydrogels. These results support the use of EHO-85 dressing, for treatment of hard-to-heal ulcers. Trial Registration AEMPS:PS/CR623/17/CE.

Long-Term Treatment and Effect of Discontinuation of Calcifediol in Postmenopausal Women with Vitamin D Deficiency: A Randomized Trial.

Vitamin D plays a major role in bone health and probably also in multiple extraskeletal acute and chronic diseases. Although supplementation with calcifediol, a vitamin D metabolite, has demonstrated efficacy and safety in short-term clinical trials, its effects after long-term monthly administration have been studied less extensively. This report describes the results of a 1-year, phase III-IV, double-blind, randomized, controlled, parallel, multicenter superiority clinical trial to assess the efficacy and safety of monthly calcifediol 0.266 mg versus cholecalciferol 25,000 IU (0.625 mg) in postmenopausal women with vitamin D deficiency (25(OH)D < 20 ng/mL). A total of 303 women were randomized and 298 evaluated. Patients were randomized 1:1:1 to calcifediol 0.266 mg/month for 12 months (Group A1), calcifediol 0.266 mg/month for 4 months followed by placebo for 8 months (Group A2), and cholecalciferol 25,000 IU/month (0.625 mg/month) for 12 months (Group B). By month 4, stable 25(OH)D levels were documented with both calcifediol and cholecalciferol (intention-to-treat population): 26.8 ± 8.5 ng/mL (Group A1) and 23.1 ± 5.4 ng/mL (Group B). By month 12, 25(OH)D levels were 23.9 ± 8.0 ng/mL (Group A1) and 22.4 ± 5.5 ng/mL (Group B). When calcifediol treatment was withdrawn in Group A2, 25(OH)D levels decreased to baseline levels (28.5 ± 8.7 ng/mL at month 4 versus 14.4 ± 6.0 ng/mL at month 12). No relevant treatment-related safety issues were reported in any of the groups. The results confirm that long-term treatment with monthly calcifediol in vitamin D-deficient patients is effective and safe. The withdrawal of treatment leads to a pronounced decrease of 25(OH)D levels. Calcifediol presented a faster onset of action compared to monthly cholecalciferol. Long-term treatment produces stable and sustained 25(OH)D concentrations with no associated safety concerns. © 2023 Faes Farma SA. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

AG5 is a potent non-steroidal anti-inflammatory and immune regulator that preserves innate immunity.

An archetypal anti-inflammatory compound against cytokine storm would inhibit it without suppressing the innate immune response. AG5, an anti-inflammatory compound, has been developed as synthetic derivative of andrographolide, which is highly absorbable and presents low toxicity. We found that the mechanism of action of AG5 is through the inhibition of caspase-1. Interestingly, we show with in vitro generated human monocyte derived dendritic cells that AG5 preserves innate immune response. AG5 minimizes inflammatory response in a mouse model of lipopolysaccharide (LPS)-induced lung injury and exhibits in vivo anti-inflammatory efficacy in the SARS-CoV-2-infected mouse model. AG5 opens up a new class of anti-inflammatories, since contrary to NSAIDs, AG5 is able to inhibit the cytokine storm, like dexamethasone, but, unlike corticosteroids, preserves adequately the innate immunity. This is critical at the early stages of any naïve infection, but particularly in SARS-CoV-2 infections. Furthermore, AG5 showed interesting antiviral activity against SARS-CoV-2 in humanized mice.

Bariatric surgery and calcifediol treatment, Gordian knot of severe-obesity-related comorbidities treatment.

Background: Obesity (OB) is a chronic metabolic disease with important associated comorbidities and mortality. Vitamin D supplementation is frequently administered after bariatric surgery (BS), so as to reduce OB-related complications, maybe including chronic inflammation. Aim: This study aimed to explore relations between vitamin D metabolites and components of the inflammasome machinery in OB before and after BS and their relations with the improvement of metabolic comorbidities. Patients and methods: Epidemiological/clinical/anthropometric/biochemical evaluation was performed in patients with OB at baseline and 6 months after BS. Evaluation of i) vitamin-D metabolites in plasma and ii) components of the inflammasome machinery and inflammatory-associated factors [NOD-like-receptors (NLRs), inflammasome-activation-components, cytokines and inflammation/apoptosis-related components, and cell-cycle and DNA-damage regulators] in peripheral blood mononuclear cells (PBMCs) was performed at baseline and 6 months after BS. Clinical and molecular correlations/associations were analyzed. Results: Significant correlations between vitamin D metabolites and inflammasome-machinery components were observed at baseline, and these correlations were significantly reduced 6 months after BS in parallel to a decrease in inflammation markers, fat mass, and body weight. Treatment with calcifediol remarkably increased 25OHD levels, despite 24,25(OH)2D3 remained stable after BS. Several inflammasome-machinery components were associated with improvement in metabolic comorbidities, especially hypertension and dyslipidemia. Conclusion: The beneficial effects of vitamin D on OB-related comorbidities after BS patients are associated with significant changes in the molecular expression of key inflammasome-machinery components. The expression profile of these inflammasome components can be dynamically modulated in PBMCs after BS and vitamin D supplementation, suggesting that this profile could likely serve as a sensor and early predictor of the reversal of OB-related complications after BS.

Influence of Dipeptidyl Peptidase-4 (DPP4) on Mesenchymal Stem-Cell (MSC) Biology: Implications for Regenerative Medicine - Review.

Dipeptidyl peptidase IV (DPP4) is a ubiquitous protease that can be found in membrane-anchored or soluble form. Incretins are one of the main DPP4 substrates. These hormones regulate glucose levels, by stimulating insulin secretion and decreasing glucagon production. Because DPP4 levels are high in diabetes, DPP4 inhibitor (DPP4i) drugs derived from gliptin are widespread used as hypoglycemic agents for its treatment. However, as DPP4 recognizes other substrates such as chemokines, growth factors and neuropeptides, pleiotropic effects have been observed in patients treated with DPP4i. Several of these substrates are part of the stem-cell niche. Thus, they may affect different physiological aspects of mesenchymal stem-cells (MSC). They include viability, differentiation, mobilization and immune response. MSC are involved in tissue homeostasis and regeneration under both physiological and pathological conditions. Therefore, such cells and their secretomes have a high clinical potential in regenerative medicine. In this context, DPP4 activity may modulate different aspects of MSC regenerative capacity. Therefore, the aim of this review is to analyze the effect of different DPP4 substrates on MSC. Likewise, how the regulation of DPP4 activity by DPP4i can be applied in regenerative medicine. That includes treatment of cardiovascular and bone pathologies, cutaneous ulcers, organ transplantation and pancreatic beta-cell regeneration, among others. Thus, DPP4i has an important clinical potential as a complement to therapeutic strategies in regenerative medicine. They involve enhancing the differentiation, immunomodulation and mobilization capacity of MSC for regenerative purposes.

Vitamin D Endocrine System and COVID-19: Treatment with Calcifediol.

The COVID-19 pandemic is the greatest challenge facing modern medicine and public health systems. The viral evolution of SARS-CoV-2, with the emergence of new variants with in-creased infectious potential, is a cause for concern. In addition, vaccination coverage remains in-sufficient worldwide. Therefore, there is a need to develop new therapeutic options, and/or to optimize the repositioning of drugs approved for other indications for COVID-19. This may include the use of calcifediol, the prohormone of the vitamin D endocrine system (VDES) as it may have potential useful effects for the treatment of COVID-19. We review the aspects associating COVID-19 with VDES and the potential use of calcifediol in COVID-19. VDES/VDR stimulation may enhance innate antiviral effector mechanisms, facilitating the induction of antimicrobial peptides/autophagy, with a critical modulatory role in the subsequent host reactive hyperinflammatory phase during COVID-19: By decreasing the cytokine/chemokine storm, regulating the renin-angiotensin-bradykinin system (RAAS), modulating neutrophil activity and maintaining the integrity of the pulmonary epithelial barrier, stimulating epithelial repair, and directly and indirectly decreasing the increased coagulability and prothrombotic tendency associated with severe COVID-19 and its complications. Available evidence suggests that VDES/VDR stimulation, while maintaining optimal serum 25OHD status, in patients with SARS-CoV-2 infection may significantly reduce the risk of acute respiratory distress syndrome (ARDS) and severe COVID-19, with possible beneficial effects on the need for mechanical ventilation and/or intensive care unit (ICU) admission, as well as deaths in the course of the disease. The pharmacokinetic and functional characteristics of calcifediol give it superiority in rapidly optimizing 25OHD levels in COVID-19. A pilot study and several observational intervention studies using high doses of calcifediol (0.532 mg on day 1 and 0.266 mg on days 3, 7, 14, 21, and 28) dramatically decreased the need for ICU admission and the mortality rate. We, therefore, propose to use calcifediol at the doses described for the rapid correction of 25OHD deficiency in all patients in the early stages of COVID-19, in association, if necessary, with the new oral antiviral agents.

Analytical platform for verification and quantitation of target peptides in human serum: application to cathelicidin.

A selective and sensitive, fully automated platform for verification and quantitative determination of target peptides in biofluids is proposed and then validated by development of a method for analysis of cathelicidin in human serum. The method is based on the on-line coupling of solid-phase extraction (SPE) and tandem mass spectrometry with direct infusion. Mass spectrometry analysis was carried out by multiple reaction monitoring using three transitions (one for quantitative analysis and two for qualitative analysis), all them confirmed by in silico fragmentation of the target peptide. Samples were prepared in the SPE workstation on a polymeric divinylbenzene resin by preconcentration, deproteinization, and cleanup, removing salts and interferences after direct injection of human serum. The analytical process required 12 min. The limits of detection and quantitation were 2.5 and 8.25 µg/L, respectively (0.20 and 0.66 pg on column). Repeatability and within-laboratory reproducibility were 2.4% and 2.7%, respectively. A dual-cartridge configuration was used to test recovery of cathelicidin in serum, resulting in 80%. Because quantitative retention in the cartridge was assessed, determination of cathelicidin was validated without using synthetic peptides labeled with stable isotopes. The hyphenated system allows full automation, thereby improving reproducibility and accuracy, as demanded by clinical analysis.

Automated targeting analysis of eicosanoid inflammation biomarkers in human serum and in the exometabolome of stem cells by SPE-LC-MS/MS.

Inflammation is a complex cascade process involved in the pathogenesis of a number of diseases or generated as response to external or internal stimuli. Current research is focused on the development of assays for fast identification and quantitation of inflammation biomarkers. Eicosanoids are the oxidation metabolites of polyunsaturated fatty acids (mainly 20-carbon fatty acids) that play a regulation role in inflammation and, therefore, they have proved to be involved in different pathological states such as cancer, atherosclerosis, arthritis and cardiovascular or immunological diseases. Eicosanoids can be metabolized by different oxygenase enzymes to prostanoids such as prostaglandins and thromboxanes or hydroxyl fatty acids such as hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids. A high-throughput automated approach is here presented for direct eicosanoid analysis in biofluids such as human serum and cells culture media. The approach is based on a hyphenated system composed by a solid-phase extraction workstation (Prospekt 2 unit) on-line coupled to a liquid chromatograph-triple quadrupole-tandem mass spectrometer. The detection limits for the target analytes ranged from 0.009 to 204 pg on-column, with precision between 2.65% and 7.33%, expressed as relative standard deviation. Accuracy studies with a dual-cartridge configuration resulted in recoveries between 78.6% and 100%, which validated internally the proposed approach ensuring highly efficient cleanup of proteins and salts. The method is reliable, robust and endowed with a great potential for implementation in clinical and routine laboratories. Analysis of culture media of stem cells stimulated with arachidonic acid was carried out to evaluate its incidence on the eicosanoid profile of the exometabolome.

Calcium citrate and vitamin D in the treatment of osteoporosis.

The combination of calcium with vitamin D (vitamin D(3) [colecalciferol]) forms the basis of preventive and therapeutic regimens for osteoporosis. A number of studies have suggested that the combination of calcium and vitamin D is effective when administered at respective dosages of at least 1200 mg and 800 IU per day, although efficacy is, as expected, affected by patient compliance. Overall, treatment with this combination appears to be effective in reducing the incidence of non-vertebral and hip fractures. Also, in all drug studies (of antiresorptive and anabolic agents and strontium ranelate) that demonstrated a reduction in risk of osteoporotic fractures, patients also took calcium and vitamin D supplements. An important finding in this regard is that vitamin D levels have been demonstrated to be inadequate in more than half of women treated for osteoporosis in the US and Europe. The capacity of the small intestine to absorb calcium salts depends on the solubility and ionization of the salts. These properties vary for different salts, with fasting calcium citrate absorption being greater than that of calcium lactogluconate and calcium carbonate. Calcium citrate formulations taken between meals may help to prevent abdominal distension and flatulence, as well as minimize the risk of renal calculus formation, thus helping to optimize patient compliance. Therefore, calcium citrate combined with vitamin D is the combination of choice for the prevention or treatment of osteoporosis.

Vitamin D Endocrine System and COVID-19.

Preclinical data strongly suggest that the vitamin D endocrine system (VDES) may have extraskeletal effects. Cells of the immune and cardiovascular systems and lungs can express the vitamin D receptor, and overall these cells respond in a coherent fashion when exposed to 1,25-dihydroxyvitamin D, the main metabolite of the VDES. Supplementation of vitamin D-deficient subjects may decrease the risk of upper respiratory infections. The VDES also has broad anti-inflammatory and anti-thrombotic effects, and other mechanisms argue for a potential beneficial effect of a good vitamin D status on acute respiratory distress syndrome, a major complication of this SARS-2/COVID-19 infection. Activation of the VDES may thus have beneficial effects on the severity of COVID-19. Meta-analysis of observational data show that a better vitamin D status decreased the requirement of intensive care treatment or decreased mortality. A pilot study in Cordoba indicated that admission to intensive care was drastically reduced by administration of a high dose of calcifediol early after hospital admission for COVID-19. A large observational study in Barcelona confirmed that such therapy significantly decreased the odds ratio (OR) of mortality (OR = 0.52). This was also the conclusion of a retrospective study in five hospitals of Southern Spain. A retrospective study on all Andalusian patients hospitalized because of COVID-19, based on real-world data from the health care system, concluded that prescription of calcifediol (hazard ratio [HR] = 0.67) or vitamin D (HR = 0.75), 15 days before hospital admission decreased mortality within the first month. In conclusion, a good vitamin D status may have beneficial effects on the course of COVID-19. This needs to be confirmed by large, randomized trials, but in the meantime, we recommend (rapid) correction of 25 hydroxyvitamin D (25OHD) deficiency in subjects exposed to this coronavirus. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.