Vitamin D3 levels in women and factors contributing to explain metabolic variations.

The elucidated metabolism of vitamin D3 in humans has been the support to explain the high involvement of this liposoluble vitamin in physiological functions. Clinical studies have associated levels of vitamin D3 metabolites with several disorders. Despite this knowledge, there is a controversy regarding the estimation of deficiency and the physiological and supraphysiological levels of vitamin D3 metabolites. The association between serum concentrations of vitamin D3 metabolites and several potentially influential factors (namely, age and anthropometric, seasonal, spatial and metabolic factors) is analyzed in this study. For this purpose, 558 women were recruited and interviewed in several Spanish provinces before blood sampling. Serum vitamin D3 and its metabolites were determined using an SPE-LC-MS/MS platform. The concentration range for vitamin D3 was 1.7-21.1 nmol/L and was influenced by body mass index (BMI), waist-to-hip ratio (WHR) and seasonal period. 25-hydroxyvitamin D3 levels were within 4.8-147.2 nmol/L and were related to WHR, season, latitude and calcium intake. The range of 24,25-dihydroxyvitamin D3, 0.3-15.0 nmol/L, was associated to BMI, WHR, season, latitude and calcium intake. Finally, energy intake influenced the vitamin D 25-hydroxylase through the 25-hydroxyvitamin D3/vitamin D3 ratio, which regulates the synthesis of the circulating form. According to these results, it is worth emphasizing the relevance of all these factors to explain the variability in serum levels of vitamin D3 and its metabolites. All these factors should be considered in future studies assessing the alteration of vitamin D3 metabolism.

Determination of glycerophospholipids in vegetable edible oils: Proof of concept to discriminate olive oil categories.

Glycerophospholipids (GPLs) constitute a chemical family within the saponifiable fraction of vegetable oils. GPLs have been scarcely studied in edible oils owing to the lack of sensitive and selective analytical methods. We have developed a method for identification, confirmation and relative quantitation of GPLs in vegetable oils. The method is based on solid-phase extraction (SPE) for isolation of GPLs and determination by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). As proof of concept, the approach has been applied to characterize GPLs in different olive oil categories, thus revealing compositional changes, which could be explained by factors such as the quality of fruits and the extraction process. Families such as glycerophosphatidic acids and phosphatidylglycerides are remarkable because of their capability to discriminate virgin olive oils from the rest of categories. These results open a door to additional studies targeted at the identification of olive oil quality by monitoring these lipids.

Evaluation of short-term storage prior to analysis of vitamin D3 and metabolites in human serum by liquid chromatography coupled to tandem mass spectrometry.

Vitamin D has been widely determined in clinical trials to elucidate its biochemical involvement in a great number of pathologies. The analysis of vitamin D and its hydroxymetabolites in biofluids such as serum or plasma is a challenging task due to limitations associated to the low concentrations of some metabolites (typically, dihydroxymetabolites), methodological interferences, and the low stability of the compounds. Among these limitations, efforts have been targeted at optimizing instrumental improvements to develop more sensitive and selective methods, while the stability of vitamin D and metabolites has not been exhaustively evaluated. In this research, several aspects regarding the short-term storage conditions of human serum have been studied to evaluate their influence on the determination of vitamin D3 and metabolites. An experimental plan has been applied to assess the influence of two relevant parameters: the storage temperature for a period of two months and the number of freeze-thaw cycles. The storage temperature affected in a different manner to vitamin D3 and its metabolites, being vitamin D3 and 1,25-dihydroxyvitamin D3 the two analytes more affected by this parameter. Concerning the freeze-thaw cycles, this variable must be limited to two cycles owing to its significant influence on the variability for quantitation of dihydroxymetabolites in human serum. Finally, lyophilization was also tested to check if serum concentrations of vitamin D3 and its metabolites were affected by this preprocessing step. The results revealed that only vitamin D3 experienced a decrease in serum concentration after two months, which does not constitute a real problem as vitamin D3 is not currently a crucial parameter to be determined in clinical trials due to its scant biological activity.

Determination of primary fatty acid amides in different biological fluids by LC-MS/MS in MRM mode with synthetic deuterated standards: Influence of biofluid matrix on sample preparation.

The recent growing interest in primary fatty acid amides (PFAMs) is due to the broad range of physiological effects they exhibit as bioindicator of pathological states. These bioactive lipids are usually in biological samples at the nanomolar level, making their detection and identification a challenging task. A method for quantitative analysis of seven main PFAMs (lauramide, myristamide, linoleamide, palmitamide, oleamide, stearamide and behenamide) in four human biofluids -namely, urine, plasma, saliva and sweat- is here reported. Two sample preparation procedures were compared to test their efficiency in each biofluid: solid-phase extraction (SPE) and protein precipitation. The latter was the best for plasma and urine, while the analysis of saliva and sweat required an SPE step for subsequent suited determination of PFAMs. Detection of the seven metabolites was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Quantitative analysis was supported on the use of stable isotopically labeled internal standards (SIL-ISs) in the calibration method, which required the synthesis of each IS from the precursor deuterated fatty acids. Detection limits for the target analytes were within 0.3-3 ng mL-1. The method was applied to a small cohort of male and female volunteers (n = 6) to estimate the relative concentration profiles in the different biofluids. The analytical features of the method supported its applicability in clinical studies aimed at elucidating the role of PFAMs metabolism.

Cultivar influence on variability in olive oil phenolic profiles determined through an extensive germplasm survey.

Despite the evident influence of the cultivar on olive oil composition, few studies have been devoted to exploring the variability of phenols in a representative number of monovarietal olive oils. In this study, oil samples from 80 cultivars selected for their impact on worldwide oil production were analyzed to compare their phenolic composition by using a method based on LC-MS/MS. Secoiridoid derivatives were the most concentrated phenols in virgin olive oil, showing high variability that was significantly due to the cultivar. Multivariate analysis allowed discrimination between four groups of cultivars through their phenolic profiles: (i) richer in aglycon isomers of oleuropein and ligstroside; (ii) richer in oleocanthal and oleacein; (iii) richer in flavonoids; and (iv) oils with balanced but reduced phenolic concentrations. Additionally, correlation analysis showed no linkage among aglycon isomers and oleocanthal/oleacein, which can be explained by the enzymatic pathways involved in the metabolism of both oleuropein and ligstroside.

Two-dimensional liquid chromatography coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3-epimer-25-monohydroxyvitamin D3.

A method based on automated on-line solid phase extraction coupled to two-dimensional liquid chromatography with tandem mass spectrometry detection (SPE-2DLC-MS/MS) is here reported for vitamin D metabolite profiling in human serum with absolute quantitation. Two-dimensional LC was configured with two complementary analytical columns, pentafluorophenyl (PFP) and C18 phases, for determination of 25 hydroxyvitamin D3 epimers and the resting bioactive metabolites of vitamin D (D3 and D2)-25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2 and 24,25-dihydroxyvitamin D3. Quantitative determination was supported on the use of a stable isotopic labelled internal standard for each analyte and the resulting method was validated by analysis of a standard reference material certified by the National Institute of Standards & Technology (NIST-972a) and 5 samples provided by the vitamin D External Quality Assurance Scheme (DEQAS). The limits of detection were between 9 and 90pg/mL for the eight analytes, and precision, expressed as relative standard deviation, was lower than 11.6%. Two-dimensional LC has shown to be the key to discriminate between 25 hydroxyvitamin D3 epimers in a quantitative analysis also involving dihydroxyvitamin D metabolites.

Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses.

The pharmacokinetics and pharmacodynamics (PK/PD) of the angiotensin-converting enzyme inhibitor (ACEI) ramiprilat after intravenous (IV) and oral (PO) administration of ramipril have not been evaluated in horses. This study was designed to establish PK profiles for ramipril and ramiprilat as well as to determine the effects of ramiprilat on serum angiotensin converting enzyme (ACE) and to select the most appropriate ramipril dose that suppresses ACE activity. Six healthy horses in a cross-over design received IV ramipril 0.050 mg/kg, PO at a dose of 0 (placebo), and 0.050, 0.10, 0.20, 0.40 and 0.80 mg/kg ramipril. Blood pressures were measured and blood samples obtained at different times. Serum ramipril and ramiprilat concentrations and serum ACE activity were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. Systemic bioavailability of ramiprilat after PO ramipril was 6-9%. Percentages of maximum ACE inhibitions from baseline were 98.88 (IV ramipril), 5.31 (placebo) and 27.68, 39.27, 46.67, 76.13 and 84.27 (the five doses of PO ramipril). Blood pressure did not change during the experiments. Although oral availability of ramiprilat was low, ramipril has sufficient enteral absorption and bioconversion to ramiprilat to induce serum ACE inhibitions of almost 85% after a dose of 0.80 mg/kg ramipril. Additional research on ramipril administration in equine patients is indicated.

Study of blood collection and sample preparation for analysis of vitamin D and its metabolites by liquid chromatography-tandem mass spectrometry.

The analysis of vitamin D status, with special emphasis on 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, is gaining interest in clinical studies due to the classical and non-classical effects attributed to this prohormone. In this research, the influence of the two steps preceding determination (viz. sample collection and preparation) on the quantitative analysis of vitamin D and its more important metabolites has been studied. Two preparation approaches, deproteination and solid-phase extraction (SPE), have been evaluated in terms of sensitivity to delimit their application, thus establishing that detection of 1,25-dihydroxyvitamin D cannot be addressed by protein precipitation. Concerning sample collection, serum and plasma reported high accuracy (above 83.3%) for vitamin D and metabolites, while precision, expressed as relative standard deviation, was below 12.9% for all analytes in both samples. Statistical analysis revealed that serum and plasma provided similar physiological levels for vitamin D3, 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3, while significantly different levels were obtained for 1,25-dihydroxyvitamin D3, always higher in plasma than in serum. Sample collection and treatment have proved to be significant in the analysis of vitamin D and its relevant metabolites.

Quantitative analytical method to evaluate the metabolism of vitamin D.

A method for quantitative analysis of vitamin D (both D2 and D3) and its main metabolites - monohydroxylated vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) and dihydroxylated metabolites (1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3) in human serum is here reported. The method is based on direct analysis of serum by an automated platform involving on-line coupling of a solid-phase extraction workstation to a liquid chromatograph-tandem mass spectrometer. Detection of the seven analytes was carried out by the selected reaction monitoring (SRM) mode, and quantitative analysis was supported on the use of stable isotopic labeled internal standards (SIL-ISs). The detection limits were between 0.3-75pg/mL for the target compounds, while precision (expressed as relative standard deviation) was below 13.0% for between-day variability. The method was externally validated according to the vitamin D External Quality Assurance Scheme (DEQAS) through the analysis of ten serum samples provided by this organism. The analytical features of the method support its applicability in nutritional and clinical studies targeted at elucidating the role of vitamin D metabolism.

Sweat: a sample with limited present applications and promising future in metabolomics.

Sweat is a biofluid with present scant use as clinical sample. This review tries to demonstrate the advantages of sweat over other biofluids such as blood or urine for routine clinical analyses and the potential when related to metabolomics. With this aim, critical discussion of sweat samplers and equipment for analysis of target compounds in this sample is made. Well established routine analyses in sweat as is that to diagnose cystic fibrosis, and the advantages and disadvantages of sweat versus urine or blood for doping control have also been discussed. Methods for analytes such as essential metals and xenometals, ethanol and electrolytes in sweat in fact constitute target metabolomics approaches or belong to any metabolomics subdiscipline such as metallomics, ionomics or xenometabolomics. The higher development of biomarkers based on genomics or proteomics as omics older than metabolomics is discussed and also the potential role of metabolomics in systems biology taking into account its emergent implementation. Normalization of the volume of sampled sweat constitutes a present unsolved shortcoming that deserves investigation. Foreseeable trends in this area are outlined.