High-pressure Liquid Chromatography-mass Spectrometry Research Articles

A negative correlation between insulin-like peptide 3 and bisphenol A in human cord blood suggests an effect of endocrine disruptors on testicular descent during fetal development.

Does a relationship exist between insulin-like peptide 3 (INSL3) and selected environmental endocrine disruptors (EEDs) in human cord blood (cb)? In the whole population (cryptorchid and control boys) cbINSL3 correlated negatively with cb free bisphenol A (BPA) providing indirect evidence for an impact of EEDs on fetal Leydig cell INSL3 production. INSL3 is a major regulator of testicular descent. This hormone has been shown to be decreased in cord blood from boys with idiopathic cryptorchidism, the most frequent male malformation. Fetal exposure to several EEDs has been suspected to be involved in the occurrence of idiopathic cryptorchidism. Correlations between cb INSL3 or testosterone and cb free bioactive BPA and maternal milk polychlorinated biphenyls (PCB153), dichlorodiphenyldichloroethylene (DDE), and monobutyl phthalate (mBP) were assessed in newborn boys in a prospective case-control study. All boys (n = 6246) born after 34 weeks of gestation were systematically screened at birth for cryptorchidism over a 3-year period (2002-2005), and a diagnosis of cryptorchidism confirmed by a senior paediatrician. We studied 52 cryptorchid (26 transient, 26 persistent) and 128 control boys born at two hospitals in southern France. INSL3 was assayed in CB by a modified validated enzyme-linked immunosorbent assay. Testosterone was measured in CB after diethyl-ether extraction by means of ultra-pressure liquid chromatography-tandem mass spectrometry. Free cbBPA was measured after an extraction step with a radioimmunoassay validated after comparison of values obtained by high-pressure liquid chromatography-mass spectrometry. The xenobiotic analysis in mothers' milk was performed after fat extraction by gas chromatography-mass spectrometry. EED concentrations were not increased in the cryptorchid versus control group although a trend for increased mBP (P = 0.09) was observed. In the whole study population, cb levels of BPA correlated negatively with INSL3 (P = 0.01; R² = 0.05) but not with testosterone. No other EED correlated with INSL3 or with testosterone. The levels of BPA and INSL3 in cb may not reflect chronic fetal exposure to EEDs. The deleterious impact of EEDs on fetal testicular descent during specific windows of development has yet to be demonstrated. The negative correlation between cb free BPA and INSL3 provides indirect evidence for an impact of EEDs on human fetal Leydig cell INSL3 production and points to cbINSL3 as a possible target of EED action during fetal testis development.

Chemical and proteolysis-derived changes during long-term storage of lactose-hydrolyzed ultrahigh-temperature (UHT) milk.

Proteolytic activity in milk may release bitter-tasting peptides and generate free amino terminals that react with carbohydrates, which initiate Maillard reaction. Ultrahigh temperature (UHT) heat treatment inactivates the majority of proteolytic enzymes in milk. In lactose-hydrolyzed milk a β-galactosidase preparation is applied to the milk after heat treatment, which has proteolytic side activities that may induce quality deterioration of long-term-stored milk. In the present study proteolysis, glycation, and volatile compound formation were investigated in conventional (100% lactose), filtered (60% lactose), and lactose-hydrolyzed (<1% lactose) UHT milk using reverse phase high-pressure liquid chromatography-mass spectrometry, proton nuclear magnetic resonance, and gas chromatography-mass spectrometry. Proteolysis was observed in all milk types. However, the degree of proteolysis was significantly higher in the lactose-hydrolyzed milk compared to the conventional and filtered milk. The proteins most prone to proteolysis were β-CN and αs1-CN, which were clearly hydrolyzed after approximately 90 days of storage in the lactose-hydrolyzed milk.

Parallel ultra high pressure liquid chromatography-mass spectrometry for the quantification of HIV protease inhibitors using dried spot sample collection format.

An assay was developed and validated for the quantification of eight protease inhibitors (indinavir (IDV), ritonavir (RTV), lopinavir (LPV), saquinavir (SQV), amprenavir (APV), nelfinavir (NFV), atazanavir (AZV) and darunavir (DRV)) in dried plasma spots using parallel ultra-high performance liquid chromatography and mass spectrometry detection in the multiple reaction monitoring mode. For each analyte an isotopically labeled internal standard was used and the assay based on liquid-solid extraction the area response ratio (analyte/IS) was found to be linear; from 0.025 μg/ml to 20 μg/ml for IDV, SQV, DRV, AZV, LPV, from 0.025 μg/ml to 10 μg/ml for NFV, APV and from 0.025 μg/ml to 5 μg/ml for RTV using 15 μl of plasma spotted on filter paper placed in a sample tube. The total analysis time was of 4 min and inter-assay accuracies and precisions were in the range of 87.7-109% and 2.5-11.8%, respectively. On dried plasma spots all analytes were found to be stable for at least 7 days. Practicability of the assay to blood was also demonstrated. The sample drying process could be reduced to 5 min using a commercial microwave system without any analyte degradation. Together with quantification, confirmatory analysis was performed on representative clinical samples.

QuEChERS and solid phase extraction methods for the determination of energy crop pesticides in soil, plant and runoff water matrices

QuEChERS and solid phase extraction (SPE) methods were applied for determining four herbicides (metazachlor, oxyfluorfen, quizalofop-p-ethyl, quinmerac) and one insecticide (α(±)-cypermethrin) in runoff water, soil, sunflower and oilseed rape plant matrices. Determination was performed using gas chromatography mass spectrometry (GC-MS), whereas high-pressure liquid chromatography mass spectrometry (HPLC-MS) was used for quinmerac. In all substrates linearity was evaluated using matrix-matched calibration samples at five concentration levels (50–1000 ng L−1 for water, 5–500 μg kg−1 for soil and 2.5–500 μg kg−1 for sunflower or oilseed rape plant). Correlation coefficient was higher than 0.992 for all pesticides in all substrates. Acceptable mean recovery values were obtained for all pesticides in water (65.4–108.8%), soil (70.0–110.0%) and plant (66.1–118.6%), with intra- and inter-day RSD% below 20%. LODs were in the range of 0.250–26.6 ng L−1 for water, 0.10–1.8 μg kg−1 for soil and 0.15–2.0 μg kg−1 for plants. The methods can be efficiently applied for field dissipation studies of the pesticides in energy crop cultivations.

Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typhimurium in response to infection-like conditions

Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Toward this end, we used single-dimension ultra-high-pressure liquid chromatography mass spectrometry to investigate the comprehensive "intact" proteome of the Gram-negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1,665 proteoforms generated by posttranslational modifications (PTMs), representing the largest microbial top-down dataset reported to date. We confirmed many previously recognized aspects of Salmonella biology and bacterial PTMs, and our analysis also revealed several additional biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions. This finding of a S-glutathionylation-to-S-cysteinylation switch in a condition-specific manner was corroborated by bottom-up proteomics data and further by changes in corresponding biosynthetic pathways under infection-like conditions and during actual infection of host cells. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents a report of S-cysteinylation in Gram-negative bacteria. Additionally, the functional relevance of these PTMs was supported by protein structure and gene deletion analyses. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.

Endocrine aspects of ovary growth and gestation in the Argentinian cockroach, Blaptica dubia (Dictyoptera, Blaberidae)

Titers of juvenile hormone (JH) III and free ecdysteroids were studied in the hemolymph of the ovoviviparous Argentinian cockroach, Blaptica dubia, related to fat body depletion and reproduction. Adult females were analyzed during the first (days 5–25) and second vitellogenic cycle (days 80–100) and during the periods of gestation. Body weight changes of adult females were closely related to ovarian growth, ootheca formation, ootheca deposition, and hatching of the nymphs. Biochemical analysis of the fat body revealed lipids as the main storage compounds, followed by glycogen, proteins, and free carbohydrates. Changes in the fat body weight and in the chemical constituents of the fat body correlated with the processes of vitellogenesis and gestation. Concentrations of JH and free ecdysteroids in the hemolymph were measured by high pressure liquid chromatography-mass spectrometry. JH III was the only JH homolog found. JH III titers were high during vitellogenesis as well as toward the end of the gestation period. Changes in the concentrations of ecdysone and 20-hydroxyecdysone were less clear. The results reveal JH III as the major gonadotropic hormone in adult females of B. dubia.

Évaluation électrophysiologique de la perception consciente. Application à la médecine d’expertise

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 102–104 cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.

New Approach for Chemometric Analysis of Mass Spectrometry Data

The search of metabolites which are present in biological samples and the comparison between different samples allow the construction of certain biochemical patterns. The mass spectrometry (MS) methodology applied to the analysis of biological samples makes it possible for the identification of many metabolites. Each obtained signal (m/z) is characteristic of a particular metabolite. However, the mass data (m/z) interpretation is difficult because of the large amount of information that they contain. In this work, we present a relatively simple tool that allows us to deal with the whole of the mass information from the chemometric analysis. The statistical analysis is a key stage in order to identify the metabolites involved in a particular biochemical pattern. We transformed the mass data matrix in a vector. By having the data as a vector, it was possible to keep all the information and also avoid the signals overlapping, which is the major problem when the total ion chromatogram (TIC) is obtained. In the approach proposed here, the mass data (m/z) matrix was split in 100 different TIC in order to avoid the signal overlapping. The 100 chromatograms were concatenated in a vector. This vector, which can be plotted as a continuous (2D pseudospectrum), greatly simplifies for one to understand the subsequent dimensional multivariate analysis. To validate the method, 19 samples from two human embryos culture medium were analyzed by high-pressure liquid chromatography-mass spectrometry (HPLC-MS). Our methodology would be applied to the obtained raw data. Later on, a multivariate analysis was conducted using a robust principal components analysis interval (robPCA) and interval partial least squares algorithm (iPLS). The results obtained allow one to differentiate the two sample populations undoubtedly, although their composition was similar.

Development of a PCR protocol to detect ochratoxin A producing moulds in food products

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 102–104 cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.

Characterization of cold-pressed and processed bergamot oils by using GC-FID, GC-MS, GC-C-IRMS, enantio-GC, MDGC, HPLC and HPLC-MS-IT-TOF

Numerous samples of Italian cold-pressed bergamot oils, representative of the industrial production of the last three seasons (2008/09, 2009/10 and 2010/11), as well as several samples of bergapten-free, concentrated and recovered oils, have been extensively investigated in the present study. The samples were analyzed to determine the composition of the volatile fraction, the oxygen heterocyclic compounds, the enantiomeric distribution of twelve volatile components and the isotopic ratios of selected volatiles. Differences from previous studies emerged. The recent improvement in cultural practices is confirmed by the increase of linalool and linalyl acetate, indicative of better quality of the essential oils. The seasonal variation of the enantiomeric excess of α-terpineol, characteristic of cold-pressed bergamot oils, is confirmed. Some oxygen heterocyclic compounds are here quantitatively determined for the first time. The presence of herniarin is confirmed in genuine samples through high-pressure liquid-chromatography–mass spectrometry–ion trap–time of flight (HPLC-MS-IT-TOF). The isotopic ratios herein reported, for all the samples analyzed, represent a useful tool for the evaluation of genuineness of cold-pressed and processed bergamot oils.

The relation between skin disorders and vitamin D

Vitamin D deficiency has been related to a high number of health disorders including skin cancer, autoimmune skin disorders, photodermatoses, atopic dermatitis and psoriasis. Special attention has been given to UVB phototherapy, as UVB is needed to convert 7‐dehydrocholesterol to previtamin D3 in the skin. When assessing vitamin D [measured as 25‐hydroxyvitamin D, 25(OH)D] in a given skin disease, attention is normally paid to the degree of insufficiency and the percentage of patients with serum 25(OH)D below 50 nmol L−1 (insufficiency). The following confounding factors should be kept in mind. (i) At what time of the year was sampling performed? In northern Europe serum 25(OH)D falls by about 1 nmol L−1 per week, corresponding to a total decrease of 25–35 nmol L−1 during winter.1, 2 (ii) Dietary supplementation habits.3 Most diets contain very little vitamin D. Oily fish contains high levels, and fish/seal liver contains very high levels. Certain food products are fortified with vitamin D, and supplementation is becoming increasingly frequent. (iii) 25(OH)D analysis method. The gold standard, high‐pressure liquid chromatography–mass spectrometry (HPLC‐MS), is expensive to perform, and other methods are most commonly used. The percentage of the study population that is 25(OH)D deficient/insufficient is very dependent on the analysis methodology.4, 5 Snellman et al.4 analysed serum 25(OH)D by three methods and found 8% insufficiency using HPLC‐MS, 22% insufficiency using a radioimmunoassay and 43% using a chemiluminescent immunoassay.

Development of a PCR Protocol To Detect Aflatoxigenic Molds in Food Products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.

A HPLC-MS method to detect and quantify guanfacine in urine

Guanfacine, an α2-adrenergic α2A)agonist long indicated to treat hypertension, is now being used to treat attention deficit-hyperactivity disorder (ADHD) in adolescents. This new therapeutic use may require urine testing to document compliance or abuse. A simple rapid high pressure liquid chromatography-mass spectrometry (HPLC-MS) method to detect and quantify guanfacine in urine following therapeutic administration is presented. Guanfacine and protriptyline internal standard were extracted from alkalinized urine with ethyl acetate. The organic layer was evaporated, reconstituted with mobile phase and analyzed on a YMC Basic S-5 micron, 2.0 × 150 mm HPLC column connected to an MS detector operated in positive electrospray ionization mode with selected ion resonance. Elution times were <5 min. The analytical measurement range for guanfacine was 20-2000 ng/mL. The limit of detection and quantitation were 5 ng/mL and 20 ng/mL, respectively. Precision as %CV was <15% at 40, 100 and 500 ng/mL (n=6). Percentage recovery using the same concentrations was >89%. Interference with drugs and biological constituents was assessed; no interferences were noted. Analysis of 100 random post-diagnostic urine specimens yielded 11 guanfacine positive results with concentrations ranging from 11 to 6390 ng/mL. This HPLC-MS method provides a simple and rapid method for the routine detection and quantitation of guanfacine in urine specimens.

Duplex real-time PCR method with internal amplification control for quantification of verrucosidin producing molds in dry-ripened foods

Verrucosidin, which is a tremorgenic mycotoxin responsible for neurological diseases, has been detected in different dry-ripened foods as consequence of the growth of toxigenic molds. To improve food safety, the presence of verrucosidin producing molds in these kind foods should be quantified. The aim of this study was to design a duplex real-time PCR (qPCR) protocol based on TaqMan methodology with an internal amplification control (IAC). Eleven verrucosidin producing and 11 non producing strains belonging to different species often reported in food products were used. Verrucosidin production was tested by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair (VerF1/VerR1) and a TaqMan probe (Verprobe) were designed from the SVr1 probe sequence of a verrucosidin producing Penicillium polonicum. The conserved regions of the β-tubulin gene were used to design primers (TubF1/TubR1) and probe (Tubprobe) of the non-competitive IAC. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves which relating Ct values and DNA template of the tested verrucosidin producers using the verrucosidin and IAC primers. The ability to quantify verrucosidin producers of the developed TaqMan assay in all artificially inoculated food samples was successful, with a minimum detection limit of 1 log cfu per gram of food. This qPCR protocol including an IAC could be very useful to quantify verrucosidin producing molds in dry-ripened foods avoiding false negative results. This method should be proposed to monitor the target molds in HACCP programs to prevent the risk of verrucosidin formation and consequently avoid its presence in the food chain.

Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the non-ribosomal peptide synthetase (otanpsPN) gene involved in OTA biosynthesis. Seventy five mold strains representing OTA producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1x10(4) to 10conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between 1 and 10conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods.

Development of a PCR protocol to detect patulin producing moulds in food products

Patulin is a secondary toxic metabolite with important health effects. Several mould species of Penicillium and Aspergillus genera associated with patulin production have been detected in food products. Thus, specific and sensitive methods to detect patulin producing moulds are needed. The aim of this work was to develop a polymerase chain reaction (PCR) method to detect patulin producing moulds in food. 34 patulin producing and 30 non-producing strains belonging to the main species usually reported in food products were used. Patulin production was firstly evaluated by mycellar electrokinetic capillary electrophoresis and high-pressure liquid chromatography-mass spectrometry in all tested strains. Biosynthesis was also used to develop PCR primers derived from the genes involved in patulin. By means of a primer pair based on the isoepoxydon dehydrogenase (idh) gene, a 496-bp amplicon was specifically detected in all the mould strains previously confirmed as patulin producing, regardless of their genus and species. With the developed method it was possible to detect down to 0.5 ng of pure DNA from producing strains and from 1.8 × 102 to 2.7 × 103 conidia g−1 in artificially inoculated foods. No relevant PCR inhibition due to food matrices was observed. The PCR protocol developed could be considered as an appropriate tool to detect patulin producing moulds in food products.

Development of real-time PCR methods to quantify patulin-producing molds in food products

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g−1 per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.

Confirmed hypertension and plasma 25(OH)D concentrations amongst elderly men

the results of experimental studies suggest that vitamin D deficiency activates the renin-angiotensin system and predisposes to hypertension. Results of previous epidemiological studies investigating the association between 25-hydroxyvitamin D [25(OH)D] status and hypertension have not been consistent, perhaps because of their sole reliance on office blood pressure (BP) measurements leading to some misclassification of hypertension status. No previous studies have examined the association between 25(OH)D status and confirmed hypertension assessed with both office and 24-h BP measurements. in this cross-sectional study, we investigated 833 Caucasian men, aged 71 ± 0.6 years, to determine the association between plasma 25(OH)D concentrations, measured with high-pressure liquid chromatography mass spectrometry, and the prevalence of hypertension. We used both supine office and 24-h BP measurements for classifying participants as normotensive or confirmed hypertensive; participants with inconsistent classifications were excluded. in a multivariable adjusted logistic regression model, men with 25(OH)D concentrations <37.5 nmol L(-1) had a 3-fold higher prevalence of confirmed hypertension compared to those with ≥ 37.5 nmol L(-1) 25(OH)D (odds ratio = 3.3, 95% CI: 1.0-11.0). our results show that low plasma 25(OH)D concentration is associated with a higher prevalence of confirmed hypertension.

Pharmacokinetics of intrarectal omeprazole in alpacas

The purpose of this study was to evaluate the pharmacokinetics of omeprazole in three different vehicles when administered rectally to six alpacas. Alpacas were given single doses of omeprazole (4 mg/kg) in a double-blinded, randomized cross-over design with a 1 week washout period. Omeprazole formulations consisted of (1) Treatment A: omeprazole paste mixed in surgical lubricant (2) Treatment B: omeprazole capsule contents in 8.4% sodium bicarbonate and (3) Treatment C: omeprazole capsule contents in surgical lubricant and 8.4% sodium bicarbonate solution. Plasma samples were drawn at 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 300 and 480 min. Omeprazole plasma concentrations were determined by high-pressure liquid chromatography-mass spectrometry. Pharmacokinetic results demonstrated median peak plasma concentrations (C(max)) of 7.35 (3.2-15.2), 7.30 (1.7-10.9) and 8.65 (1.8-19.3) ng/mL and median area under the concentration curve (AUC((0-180))) of 747 (237-1681) min x ng/mL, 552.9 (39-1063) min x ng/mL, and 972 (107-1841) min x ng/mL for treatments A, B and C, respectively. The median half-lives were similar between groups: 38, 50, and 53 min. As a result of the low measured omeprazole plasma concentrations, it is assumed that rectal absorption of omeprazole is poor in alpacas and not an effective route of administration.

Plasma 25-Hydroxyvitamin D Levels and Fracture Risk in a Community-Based Cohort of Elderly Men in Sweden

Blood levels of 25-hydroxyvitamin D [25(OH)D] is the generally accepted indicator of vitamin D status, but no universal reference level has been reached. The objective of the study was to determine the threshold at which low plasma 25(OH)D levels are associated with fractures in elderly men and clarify the importance of low levels on total fracture burden. In the Uppsala Longitudinal Study of Adult Men, a population-based cohort (mean age, 71 yr, n = 1194), we examined the relationship between 25(OH)D and risk for fracture. Plasma 25(OH)D levels were measured with high-pressure liquid chromatography-mass spectrometry. The study was conducted in the municipality of Uppsala in Sweden, a country with a high fracture incidence. Time to fracture was measured. During follow-up (median 11 yr), 309 of the participants (26%) sustained a fracture. 25(OH)D levels below 40 nmol/liter, which corresponded to the fifth percentile of 25(OH)D, were associated with a modestly increased risk for fracture, multivariable-adjusted hazard ratio 1.65 (95% confidence interval 1.09-2.49). No risk difference was detected above this level. Approximately 3% of the fractures were attributable to low 25(OH)D levels in this population. Vitamin D insufficiency is not a major cause of fractures in community-dwelling elderly men in Sweden. Despite the fact that cutaneous synthesis of previtamin D during the winter season is undetectable at this northern latitude of 60 degrees, only one in 20 had 25(OH)D levels below 40 nmol/liter, the threshold at which the risk for fracture started to increase. Genetic adaptations to limited UV light may be an explanation for our findings.