Nestekromatografia-tandem massaspektrometria steroidien ja steroidiglukuroni konjugaattien tutkimisessa aivo- ja virtsanäytteistä

Steroids are endogenous compounds, which are present in the body tissues and fluids in free and conjugated forms, e.g. as steroid glucuronide conjugates. First goal of the study was to develop methodologies for the synthesis of steroid glucuronides. An enzyme-assisted synthesis was carried out to produce glucuronide-conjugated steroids at milligram levels. Hepatic microsomal preparations of bovine, porcine, and Arochlor-induced rats were compared with respect to specificity and efficiency of uridine diphosphate glucuronosyl transferase (UGT) enzymes in steroid glucuronidation. Both bovine and porcine liver microsomes efficiently produced the steroid glucuronides of all eight steroids studied, whereas rat liver microsomes produced glucuronides efficiently only for three steroids. Synthesized steroid glucuronides were purified with solid-phase extraction (SPE) or LC fractionation and characterized by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution MS. Steroid glucuronides were obtained in milligram amounts with good yields. The synthesised glucuronides were used in method development and as reference material in the analysis. As a second goal of the study, two methods were developed, one for brain samples using capillary liquid chromatography-electrospray-tandem mass spectrometry (CapLC-ESI-MS/MS) and the other for the urine samples using ultra performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS).The methods were carefully optimized to obtain good selectivity and maximum sensitivity. Linear range of 3-4 magnitudes was obtained (R2> 0.996) with good precision (RSD < 15%). Detection limits of 6-100 pmol/l were obtained for steroid glucuronides, 10-30 pmol/l for steroid sulphates and 0.03-22 nmol/l for free steroids, respectively. The developed methods were applied to the analysis of mouse brain extracts, to study in vitro metabolism of steroids in rat and mouse brain, and to the analysis of free and glucuronide conjugated steroids during pregnancy. Using the CapLC-MS/MS method steroid glucuronides were observed for the first time in brain. The UPLC-MS/MS method employed in determination of urinary profiles of steroids and steroid glucuronide conjugates steroids during pregnancy. The concentrations of 36 targeted steroids and steroid glucuronides as well as the mostly unidentified C21-steroid glucuronides clearly altered during the pregnancy. In general, the concentrations of the steroids and steroid glucuronides gradually increased during pregnancy, decreased rapidly just before or during delivery, and returned to control sample level five days after the delivery.Steroidi hormonit ovat elimistön omia yhdisteitä, jotka esiintyvät elimistön kudoksissa ja nesteissä vapaassa muodossa, sitoutuneena proteiineihin sekä konjugoituina esim. steroidi glukuroni konjugaatteina. Tutkimuksen ensimmäinen tavoite oli kehittää entsyymiavusteisia synteesimenetelmiä steroidi glukuronien tuottamiseksi vertailuaineeksi menetelmän kehitykseen. Uridiini difosfaatti glucurononyylitransferaasi (UGT) entsyymien spesifisyyttä ja tehoa vertailtiin naudan, sian ja rotan maksan mikrosomi-preparaatteissa. Naudan ja sian maksa tuottivat tehokkaasti synteesituotteet kaikille kahdeksalle tutkitulle steroidille, kun taas rotan maksa tuotti tehokkaasti synteesituotteet vain kolmelle tuotteelle. Synteesituotteet puhdistettiin ja karakterisoitiin. Puhdistettuja steroidi glukuroneja pystyttiin tuottamaan milligramma määriä hyvällä puhtausasteella (>85 %). Tuotettuja steroidi glukuroneja käytettiin menetelmän kehityksessä ja menetelmien vertailuaineina. Tutkimuksen toinen tavoite oli menetelmän kehitys steroideille ja steroidi glukuroni konjugaateille biologisista matriiseista. Yksi menetelmä kehitettiin aivonäyteille käyttäen kapillaari nestekromatografia-tandem massaspektrometriaa (CapLC-MS/MS) ja toinen virtsanäytteille käyttäen korkean suorituskyvyn kromatografia -tandem massaspektrometriaa (UPLC-MS/MS) hyödyntäen. Menetelmät optimointiin ja validointiin. Menetelmät havaittiin soveltuviksi käyttötarkoituksiinsa. Menetelmien havaintorajat olivat 6-100 pmol/l steroid glukuronideille, 10-30 pmol/l steroidi sulfaateille ja 0.03-22 nmol/l vapaille steroideille. Kehitettyjä menetelmiä sovellettiin hiiren aivojen analyyseissä, in vitro metabolian tutkimiseen rotan ja hiiren aivoista valmistetuista S9 preparaateissa ja raskauden aikaisten virtsanäytteiden tutkimiseen. CapLC-MS/MS menetelmää hyödyntäen voitiin osoittaa ensimmäistä kertaa maailmassa steroidi glukuronien esiintyminen aivoissa. UPLC-MS/MS menetelmää hyödyntäen seurattiin steroidi ja steroidi glukuroni profiilien muuttumista raskauden aikana. Kolmenkymmenenkuuden tunnetun ja tuntemattoman steroidin ja steroidiglukuronin pitoisuus muuttui selvästä raskauden edetessä. Yleisesti, pitoisuudet nousivat asteittain heti raskauden alkamisen jälkeen ja pienenivät juuri ennen synnytystä tai synnytyksen aikana, ja palasivat kontrollinäytteen tasolle viisi päivää synnytyksen jälkeen

Targeted Clinical Metabolite Profiling Platform for the Stratification of Diabetic Patients

Several small molecule biomarkers have been reported in the literature for prediction and diagnosis of (pre)diabetes, its co-morbidities, and complications. Here, we report the development and validation of a novel, quantitative method for the determination of a selected panel of 34 metabolite biomarkers from human plasma. We selected a panel of metabolites indicative of various clinically-relevant pathogenic stages of diabetes. We combined these candidate biomarkers into a single ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and optimized it, prioritizing simplicity of sample preparation and time needed for analysis, enabling high-throughput analysis in clinical laboratory settings. We validated the method in terms of limits of detection (LOD) and quantitation (LOQ), linearity (R2), and intra- and inter-day repeatability of each metabolite. The method’s performance was demonstrated in the analysis of selected samples from a diabetes cohort study. Metabolite levels were associated with clinical measurements and kidney complications in type 1 diabetes (T1D) patients. Specifically, both amino acids and amino acid-related analytes, as well as specific bile acids, were associated with macro-albuminuria. Additionally, specific bile acids were associated with glycemic control, anti-hypertensive medication, statin medication, and clinical lipid measurements. The developed analytical method is suitable for robust determination of selected plasma metabolites in the diabetes clinic

Exposure to environmental contaminants is associated with altered hepatic lipid metabolism in non-alcoholic fatty liver disease

Background & aims: Recent experimental models and epidemiological studies suggest that specific environmental contaminants (ECs) contribute to the initiation and pathology of nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms linking EC exposure with NAFLD remain poorly understood and there is no data on their impact on the human liver metabolome. Herein, we hypothesized that exposure to ECs, particularly perfluorinated alkyl substances (PFAS), impacts liver metabolism, specifically bile acid metabolism. Methods: In a well-characterized human NAFLD cohort of 105 individuals, we investigated the effects of EC exposure on liver metabolism. We characterized the liver (via biopsy) and circulating metabolomes using 4 mass spectrometry-based analytical platforms, and measured PFAS and other ECs in serum. We subsequently compared these results with an exposure study in a PPARa-humanized mouse model. Results: PFAS exposure appears associated with perturbation of key hepatic metabolic pathways previously found altered in NAFLD, particularly those related to bile acid and lipid metabolism. We identified stronger associations between the liver metabolome, chemical exposure and NAFLD-associated clinical variables (liver fat content, HOMA-IR), in females than males. Specifically, we observed PFAS-associated upregulation of bile acids, triacylglycerols and ceramides, and association between chemical exposure and dysregulated glucose metabolism in females. The murine exposure study further corroborated our findings, vis-a-vis a sex-specific association between PFAS exposure and NAFLD-associated lipid changes. Conclusions: Females may be more sensitive to the harmful impacts of PFAS. Lipid-related changes subsequent to PFAS exposure may be secondary to the interplay between PFAS and bile acid metabolism. Lay summary: There is increasing evidence that specific environmental contaminants, such as perfluorinated alkyl substances (PFAS), contribute to the progression of non-alcoholic fatty liver disease (NAFLD). However, it is poorly understood how these chemicals impact human liver metabolism. Here we show that human exposure to PFAS impacts metabolic processes associated with NAFLD, and that the effect is different in females and males. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.Peer reviewe

Genome-scale study reveals reduced metabolic adaptability in patients with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a major risk factor leading to chronic liver disease and type 2 diabetes. Here we chart liver metabolic activity and functionality in NAFLD by integrating global transcriptomic data, from human liver biopsies, and metabolic flux data, measured across the human splanchnic vascular bed, within a genome-scale model of human metabolism. We show that an increased amount of liver fat induces mitochondrial metabolism, lipolysis, glyceroneogenesis and a switch from lactate to glycerol as substrate for gluconeogenesis, indicating an intricate balance of exacerbated opposite metabolic processes in glycemic regulation. These changes were associated with reduced metabolic adaptability on a network level in the sense that liver fat accumulation puts increasing demands on the liver to adaptively regulate metabolic responses to maintain basic liver functions. We propose that failure to meet excessive metabolic challenges coupled with reduced metabolic adaptability may lead to a vicious pathogenic cycle leading to the co-morbidities of NAFLD.Peer reviewe

Conjugated C-6 hydroxylated bile acids in serum relate to human metabolic health and gut Clostridia species

Knowledge about in vivo effects of human circulating C-6 hydroxylated bile acids (BAs), also called muricholic acids, is sparse. It is unsettled if the gut microbiome might contribute to their biosynthesis. Here, we measured a range of serum BAs and related them to markers of human metabolic health and the gut microbiome. We examined 283 non-obese and obese Danish adults from the MetaHit study. Fasting concentrations of serum BAs were quantified using ultra-performance liquid chromatography-tandem mass-spectrometry. The gut microbiome was characterized with shotgun metagenomic sequencing and genome-scale metabolic modeling. We find that tauro- and glycohyocholic acid correlated inversely with body mass index (P = 4.1e-03, P = 1.9e-05, respectively), waist circumference (P = 0.017, P = 1.1e-04, respectively), body fat percentage (P = 2.5e-03, P = 2.3e-06, respectively), insulin resistance (P = 0.051, P = 4.6e-4, respectively), fasting concentrations of triglycerides (P = 0.06, P = 9.2e-4, respectively) and leptin (P = 0.067, P = 9.2e-4). Tauro- and glycohyocholic acids, and tauro-a-muricholic acid were directly linked with a distinct gut microbial community primarily composed of Clostridia species (P = 0.037, P = 0.013, P = 0.027, respectively). We conclude that serum conjugated C-6-hydroxylated BAs associate with measures of human metabolic health and gut communities of Clostridia species. The findings merit preclinical interventions and human feasibility studies to explore the therapeutic potential of these BAs in obesity and type 2 diabetes.Peer reviewe

Exposure to environmental contaminants is associated with altered hepatic lipid metabolism in non-alcoholic fatty liver disease

Background & aimsRecent experimental models and epidemiological studies suggest that specific environmental contaminants (ECs) contribute to the initiation and pathology of NAFLD. However, the underlying mechanisms linking EC exposure with NAFLD remain poorly understood and there is no data on their impact on the human liver metabolome. Herein, we hypothesized that exposure to ECs, particularly perfluorinated alkyl substances (PFAS), impacts liver metabolism, specifically bile acid metabolism.MethodsIn a well-characterized human NAFLD cohort of 105 individuals, we investigated the effects of EC exposure on liver metabolism. We characterized the liver (via biopsy) and circulating metabolomes using four mass spectrometry-based analytical platforms, and measured PFAS and other ECs in serum. We subsequently compared these results with an exposure study in a PPARα-humanized mouse model.ResultsPFAS exposure appears associated with perturbation of key hepatic metabolic pathways previously found altered in NAFLD, particularly as regards bile acid and lipid metabolism. We identified stronger associations between the liver metabolome, chemical exposure and NAFLD-associated clinical variables (liver fat content, HOMA-IR), in female subjects versus males. Specifically, we observed PFAS-associated up-regulation of bile acids, triacylglycerols and ceramides, and association between chemical exposure and dysregulated glucose metabolism in females. The murine exposure study further corroborated our findings, vis-à-vis a sex-specific association between PFAS exposure and NAFLD-associated lipid changes.ConclusionsFemales may be more sensitive to the harmful impacts of PFAS. Lipid-related changes subsequent to PFAS exposure may be secondary to the interplay between PFAS and bile acid metabolism.</p

Human Serum Metabolites Associate With Severity and Patient Outcomes in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide, especially in children and young adults. TBI is an example of a medical condition where there are still major lacks in diagnostics and outcome prediction. Here we apply comprehensive metabolic profiling of serum samples from TBI patients and controls in two independent cohorts. The discovery study included 144 TBI patients, with the samples taken at the time of hospitalization. The patients were diagnosed as severe (sTBI; n=22), moderate (moTBI; n=14) or mild TBI (mTBI; n=108) according to Glasgow Coma Scale. The control group (n=28) comprised of acute orthopedic non-brain injuries. The validation study included sTBI (n=23), moTBI (n=7), mTBI (n=37) patients and controls (n=27). We show that two medium-chain fatty acids (decanoic and octanoic acids) and sugar derivatives including 2,3-bisphosphoglyceric acid are strongly associated with severity of TBI, and most of them are also detected at high concentrations in brain microdialysates of TBI patients. Based on metabolite concentrations from TBI patients at the time of hospitalization, an algorithm was developed that accurately predicted the patient outcomes (AUC=0.84 in validation cohort). Addition of the metabolites to the established clinical model (CRASH), comprising clinical and computed tomography data, significantly improved prediction of patient outcomes. The identified 'TBI metabotype' in serum, that may be indicative of disrupted blood-brain barrier, of protective physiological response and altered metabolism due to head trauma, offers a new avenue for the development of diagnostic and prognostic markers of broad spectrum of TBIs.European Union FP7 project TBIcare (Grant ID: 270259), GE-NFL Head Health Challenge I Award (Grant ID: 7620), EVO (Finland), Maire Taponen Foundation, National Institute for Health Research, National Institute for Health Research Biomedical Research Centre Cambridge (Neuroscience Theme; Brain Injury and Repair Theme)This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.ebiom.2016.07.01

Conjugated C-6 hydroxylated bile acids in serum relate to human metabolic health and gut Clostridia species

Knowledge about in vivo effects of human circulating C-6 hydroxylated bile acids (BAs), also called muricholic acids, is sparse. It is unsettled if the gut microbiome might contribute to their biosynthesis. Here, we measured a range of serum BAs and related them to markers of human metabolic health and the gut microbiome. We examined 283 non-obese and obese Danish adults from the MetaHit study. Fasting concentrations of serum BAs were quantified using ultra-performance liquid chromatography-tandem mass-spectrometry. The gut microbiome was characterized with shotgun metagenomic sequencing and genome-scale metabolic modeling. We find that tauro- and glycohyocholic acid correlated inversely with body mass index (P = 4.1e-03, P = 1.9e-05, respectively), waist circumference (P = 0.017, P = 1.1e-04, respectively), body fat percentage (P = 2.5e-03, P = 2.3e-06, respectively), insulin resistance (P = 0.051, P = 4.6e-4, respectively), fasting concentrations of triglycerides (P = 0.06, P = 9.2e-4, respectively) and leptin (P = 0.067, P = 9.2e-4). Tauro- and glycohyocholic acids, and tauro-a-muricholic acid were directly linked with a distinct gut microbial community primarily composed of Clostridia species (P = 0.037, P = 0.013, P = 0.027, respectively). We conclude that serum conjugated C-6-hydroxylated BAs associate with measures of human metabolic health and gut communities of Clostridia species. The findings merit preclinical interventions and human feasibility studies to explore the therapeutic potential of these BAs in obesity and type 2 diabetes.</p