Sterols and oxysterols

The ingredients of animal feed formulation, especially the fatty sources, play a significant role in the production of high-quality food of animal origin. Industrial fat by-products are a major source of feeding fats in Europe. The main objectives of the project were firstly to assess feed fats and oils for their composition and content of sterols (cholesterol and phytosterols) and oxysterols, and secondly to assess the levels of cholesterol and oxycholesterols of chicken and rabbit tissues after feeding with especially formulated feeds containing trans fatty acids and oxidized lipids. The lack of standardized analytical procedure prompted the evaluation of an in-house purification method by comparing it with a number of commonly used methods for the analysis of oxysterols. The saponification and transesterification steps showed rather comparable results. A two-dimensional capillary GC column with different stationary phases (a 35% phenyl column coupled to an apolar 5% phenyl column) was used for better resolution of a large number of oxysterols. This new system improved the separation efficiency and reduced the analytical time for a wide range of oxysterols. The satisfactory purification method and the reliable separation of oxysterols facilitated the qualitative and quantitative assessment of sterols and oxysterols in samples of by-products from chemical and physical refining. A large variation in the levels of sterols and oxysterols was observed in the fat by-products from chemical and physical refining processes for edible fats and oils. The observed variations in the contents and composition of sterols and oxysterols were mainly due to the origins, production facility and different processing conditions of the by-product samples. The highly oxidized lipid and trans fatty acid feeds significantly increased the contents of cholesterol and oxycholesterols in edible parts, e.g. the muscles and livers of chickens and rabbits (0.01< p ≤0.05). Hence, the consumption of products from animals fed with higher levels of trans fatty acids and oxidized lipid feeds may contribute to higher ingestion of cholesterol and oxycholesterols by humans

Concentrations of canine prostate specific esterase, CPSE, at baseline are associated with the relative size of the prostate at three-year follow-up

BackgroundEnlargement of the prostate is associated with prostatic diseases in dogs, and an estimation of prostatic size is a central part in the diagnostic workup. Ultrasonography is often the method of choice, but biomarkers constitute an alternative. Canine prostate specific esterase (CPSE) shares many characteristics with human prostate specific antigen (PSA) and is related to prostate size. In men with clinical symptoms of prostatic disease, PSA concentrations are related to prostate growth. The aims of the present follow-up study were to evaluate if the concentration of CPSE is associated with future growth of the prostate, and if analysis of a panel of 16 steroids gives further information on prostatic growth. Owners of dogs included in a previous study were 3 years later contacted for a follow-up study that included an interview and a clinical examination. The prostate was examined by ultrasonography. Serum concentrations of CPSE were measured, as was a panel of steroids.ResultsOf the 79 dogs included at baseline, owners of 77 dogs (97%) were reached for an interview, and 22 were available for a follow-up examination. Six of the 79 dogs had clinical signs of prostatic disease at baseline, and eight of the remaining 73 dogs (11%) developed clinical signs between baseline and follow-up, information was lacking for two dogs. Development of clinical signs was significantly more common in dogs with a relative prostate size of >= 2.5 at baseline (n=20) than in dogs with smaller prostates (n=51). Serum concentrations of CPSE at baseline were not associated with the change in prostatic size between baseline and follow-up. Serum concentrations of CPSE at baseline and at follow-up were positively associated with the relative prostatic size (S-rel) at follow-up. Concentrations of corticosterone (P = 0.024), and the class corticosteroids (P = 0.0035) were positively associated with the difference in S-rel between baseline and follow-up.ConclusionsThe results support the use of CPSE for estimating present and future prostatic size in dogs >= 4years, and the clinical usefulness of prostatic size for predicting development of clinical signs of prostatic disease in the dog. The association between corticosteroids and prostate growth warrants further investigation

Analysis of tryptophan metabolites and related compounds in human and murine tissue : development and validation of a quantitative and semi-quantitative method using high resolution mass spectrometry

This study explores the metabolic differences between human and murine plasma in addition to differences between murine subcutaneous and visceral white adipose tissue. A quantitative and semi-quantitative targeted method was developed and validated for this purpose. The quantitative method includes tryptophan and its metabolites in addition to tyrosine, phenylalanine, taurine, B vitamins, neopterin, cystathionine and hypoxanthine. While the semi-quantitative method includes; 3-indoleacetic acid, 5-hydroxyindoleacetic acid, acetylcholine, asymmetric dimethylarginine, citrulline and methionine. Sample preparation was based on protein precipitation, while quantification was conducted using ultrahigh-performance liquid chromatography coupled to a quadrupole Orbitrap tandem mass spectrometer with electrospray ionization in the parallel reaction monitoring (PRM) mode. The low limit of quantification for all metabolites ranged from 1 to 200 ng mL-1. Matrix effects and recoveries for stable isotope labelled internal standards were evaluated, with most having a coefficient of variation (CV) of less than 15%. Results showed that a majority of the analytes passed both the intra- and interday precision and accuracy criteria. The comparative analysis of human and murine plasma metabolites reveals species-specific variations within the tryptophan metabolic pathway. Notably, murine plasma generally exhibits elevated concentrations of most compounds in this pathway, with the exceptions of kynurenine and quinolinic acid. Moreover, the investigation uncovers noteworthy metabolic disparities between murine visceral and subcutaneous white adipose tissues, with the subcutaneous tissue demonstrating significantly higher concentrations of tryptophan, phenylalanine, tyrosine, and serotonin. The findings also show that even a semi-quantitative method can provide comparable results to quantitative methods from other studies and be effective for assessing metabolites in a complex sample. Overall, this study provides a robust platform to compare human and murine metabolism, providing a valuable insight to future investigations. A validated HRMS method for measuring tryptophan metabolites and related compounds has been developed, with simple sample preparation, successfully applied in human and murine plasma, as well as murine white adipose tissue

A Novel Targeted Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid Chromatography Hyphenated to Tandem Mass Spectrometry

Ultra-performance supercritical fluid chromatography-tandem mass spectrometry (UPSFC-MS/MS) is an alternative method for steroid analysis. Continuous development of analytical methodologies for steroid profiling is of major importance in the clinical environment to provide useful and more comprehensive data. The aim of this study was to identify and quantify a large number of endogenous steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) simultaneously within a short analytical time. This novel UPSFC-MS/MS method with electrospray in positive ionisation (ESI+) mode is robust, selective and present sufficiently high sensitivity to profile nineteen steroids in 50 mu L human plasma. Under optimised conditions, nineteen different steroids were separated with high efficiency in the multiple reaction monitoring (MRM) mode. The linearity of the method was good with correlation coefficients (R-2) in the range of 0.9983-0.9999 and with calibration range from 0.05-500 ng/mL in human plasma. The intraday and interday precision of the method, as RSD, was less than 15%. The accuracy of the nineteen analytes varied between 80 to 116%. Finally, the novel method was successfully applied for the determination of nineteen steroids within 5 minutes providing the possibility to use it for research as well as routine healthcare practice

Hyperandrogenic Symptoms Are a Persistent Suffering in Midlife Women with PCOS; a Prospective Cohort Study in Sweden

Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women, and the majority suffers from hyperandrogenism. Hyperandrogenism causes psychological morbidity and impaired quality of life in women with PCOS during the reproductive years, but data on prevalence and impact during midlife are lacking. Thus, this study aimed to address whether hyperandrogenism persists into midlife and, if so, what impact it has on quality of life. In order to answer this question, we performed a multicenter prospective cohort study, where we included women already diagnosed with PCOS who had reached the age of 45 years or more and age-matched controls. All participants underwent a physical exam, structured medical interview, biochemical testing and filled out self-assessment questionnaires. More than 40% of the women with PCOS and 82% of those who presented with the hyperandrogenic phenotype at the diagnostic work-up still suffered from hirsutism. Circulating testosterone levels were similar between women with PCOS and controls while free androgen index was higher in women with PCOS, independent of weight. Women with hyperandrogenic PCOS expressed persisting concerns regarding hirsutism at the follow-up assessment. In conclusion, women with PCOS who present with hyperandrogenic symptoms at the time they are diagnosed with PCOS have a higher risk of persistent androgenic symptoms and impaired quality of life in midlife

Hyperandrogenic symptoms are a persistent suffering in midlife women with pcos; a prospective cohort study in sweden

Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women, and the majority suffers from hyperandrogenism. Hyperandrogenism causes psychological morbidity and impaired quality of life in women with PCOS during the reproductive years, but data on prevalence and impact during midlife are lacking. Thus, this study aimed to address whether hyperandrogenism persists into midlife and, if so, what impact it has on quality of life. In order to answer this question, we performed a multicenter prospective cohort study, where we included women already diagnosed with PCOS who had reached the age of 45 years or more and age-matched controls. All participants underwent a physical exam, structured medical interview, biochemical testing and filled out self-assessment questionnaires. More than 40% of the women with PCOS and 82% of those who presented with the hyperandrogenic phenotype at the diagnostic work-up still suffered from hirsutism. Circulating testosterone levels were similar between women with PCOS and controls while free androgen index was higher in women with PCOS, independent of weight. Women with hyperandrogenic PCOS expressed persisting concerns regarding hirsutism at the follow-up assessment. In conclusion, women with PCOS who present with hyperandrogenic symptoms at the time they are diagnosed with PCOS have a higher risk of persistent androgenic symptoms and impaired quality of life in midlife

Domestication Effects on Stress Induced Steroid Secretion and Adrenal Gene Expression in Chickens

Understanding the genetic basis of phenotypic diversity is a challenge in contemporary biology. Domestication provides a model for unravelling aspects of the genetic basis of stress sensitivity. The ancestral Red Junglefowl (RJF) exhibits greater fear-related behaviour and a more pronounced HPA-axis reactivity than its domesticated counterpart, the White Leghorn (WL). By comparing hormones (plasmatic) and adrenal global gene transcription profiles between WL and RJF in response to an acute stress event, we investigated the molecular basis for the altered physiological stress responsiveness in domesticated chickens. Basal levels of pregnenolone and dehydroepiandrosterone as well as corticosterone response were lower in WL. Microarray analysis of gene expression in adrenal glands showed a significant breed effect in a large number of transcripts with over-representation of genes in the channel activity pathway. The expression of the best-known steroidogenesis genes were similar across the breeds used. Transcription levels of acute stress response genes such as StAR, CH25 and POMC were upregulated in response to acute stress. Dampened HPA reactivity in domesticated chickens was associated with changes in the expression of several genes that presents potentially minor regulatory effects rather than by means of change in expression of critical steroidogenic genes in the adrenal.Funding agencies: Swedish Research Council (VR) [621-2011-4731]; Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) [221-2011-1088]; SRC [621-2011-5523]; ERC [322206]; Swedish Centre of Excellence in Animal Welfare</p

Functional properties of low-modulus PMMA bone cements containing linoleic acid

Acrylic bone cements modified with linoleic acid are a promising low-modulus alternative to traditional high-modulus bone cements. However, several key properties remain unexplored, including the effect of autoclave sterilization and the potential use of low-modulus cements in other applications than vertebral augmentation. In this work, we evaluate the effect of sterilization on the structure and stability of linoleic acid, as well as in the handling properties, glass transition temperature, mechanical properties, and screw augmentation potential of low-modulus cement containing the fatty acid. Neither 1H NMR nor SFC-MS/MS analysis showed any detectable differences in autoclaved linoleic acid compared to fresh one. The peak polymerization temperature of the low-modulus cement was much lower (28–30 °C) than that of the high-modulus cement (67 °C), whereas the setting time remained comparable (20–25 min). The Tg of the low-modulus cement was lower (75–78 °C) than that of the high-stiffness cement (103 °C). It was shown that sterilization of linoleic acid by autoclaving did not significantly affect the functional properties of low-modulus PMMA bone cement, making the component suitable for sterile production. Ultimately, the low-modulus cement exhibited handling and mechanical properties that more closely match those of osteoporotic vertebral bone with a screw holding capacity of under 2000 N, making it a promising alternative for use in combination with orthopedic hardware in applications where high-stiffness augmentation materials can result in undesired effects.Authors in thesis list of papers: C. Robo, D. Wenner, M. Nilsson, J. Hilborn, C. Öhman-Mägi, C. Persson</p

A fast ultra performance supercritical fluid chromatography-tandem mass spectrometric method for profiling of targeted phytosterols

Phytosterols are essential structural components of plant cell membranes and possess health-related benefits, including lowering blood cholesterol levels in humans. Numerous analytical methods are being used to profile plant and animal sterols. Chromatography hyphenated to tandem mass spectrometry, is a better option due to its specificity, selectivity, and sensitivity. An ultra-performance supercritical fluid chromatography hyphenated with atmospheric pressure chemical ionization (APCI) tandem mass spectrometric method was developed and eval-uated for fingerprint analysis of seven phytosterols. Mass spectrometry fragmentation behavior was used for phytosterol identification, and multiple reaction monitoring scanning was utilized for phytosterol confirmation, where APCI outperformed superiority in terms of ion intensity, particularly in the production of [M + H-H2O]+ ions rather than [M + H]+ ions. The chromatographic conditions were thoroughly evaluated, and the ionization parameters were optimized as well. In a 3 min. run, the seven phytosterols were separated concurrently. The calibration and repeatability tests were conducted to check the instrument's performance, and the results indicated that all of the phytosterols tested had correlation coefficients (r2) greater than 0.9911 over the con-centration range of 5-5000 ng/mL. The limit of quantification was below 20 ng/mL for all the tested analytes except for stigmasterol and campesterol. The partially validated method was applied for the evaluation of phytosterols in pure coconut oil and palm oil in order to demonstrate its applicability. Total sterols in coconut and palm oils were 126.77 ng/mL and 101.73 ng/mL, respectively. In comparison to earlier methods of phytosterol analysis, the novel method offers a far faster, more sensitive, and more selective analytical process

Effects of the domestic thyroid stimulating hormone receptor (TSHR) variant on the hypothalamic-pituitary-thyroid axis and behavior in chicken

Domestic chickens are less fearful, have a faster sexual development, grow bigger, and lay more eggs than their primary ancestor, the red junglefowl. Several candidate genetic variants selected during domestication have been identified, but only a few studies have directly linked them with distinct phenotypic traits. Notably, a variant of the thyroid stimulating hormone receptor (TSHR) gene has been under strong positive selection over the past millennium, but its function and mechanisms of action are still largely unresolved. We therefore assessed the abundance of the domestic TSHR variant and possible genomic selection signatures in an extensive data set comprising multiple commercial and village chicken populations as well as wild-living extant members of the genus Gallus. Furthermore, by mean of extensive backcrossing we introgressed the wild-type TSHR variant from red junglefowl into domestic White Leghorn chickens and investigated gene expression, hormone levels, cold adaptation, and behavior in chickens possessing either the wild-type or domestic TSHR variant. While the domestic TSHR was the most common variant in all studied domestic populations and in one of two red junglefowl population, it was not detected in the other Gallus species. Functionally, the individuals with the domestic TSHR variant had a lower expression of the TSHR in the hypothalamus and marginally higher in the thyroid gland than wild-type TSHR individuals. Expression of TSHB and DIO2, two regulators of sexual maturity and reproduction in birds, was higher in the pituitary gland of the domestic-variant chickens. Furthermore, the domestic variant was associated with higher activity in the open field test. Our findings confirm that the spread of the domestic TSHR variant is limited to domesticated chickens, and to a lesser extent, their wild counterpart, the red junglefowl. Furthermore, we showed that effects of genetic variability in TSHR mirror key differences in gene expression and behavior previously described between the red junglefowl and domestic chicken.Funding Agencies|Swedish Research Council (VR)Swedish Research Council [2015-05444, 2015-4870, 2017-06218]; European Research Council (ERC)European Research Council (ERC)European Commission [322206 GENEWELL]</p