Method development for the analysis of steroids, vitamin D and oxysterols

Steroids, vitamin D and oxysterols are all part of a group of compounds called lipids, which are generally important in the function of the human body and in many cases the monitoring of these analytes can be of high value. Therefore, the development of a sensitive and universal method, suitable for the simultaneous analysis of these compounds, is of a great importance. In this study, the goal was to develop and test new mass spectrometry based methods for analyzing steroids, vitamin D and oxysterols. First, the feasibility of capillary liquid chromatography-microchip atmospheric pressure photoionization-tandem mass spectrometry was examined for the analysis of anabolic steroids in human urine. The results show that this microchip based method can be feasible for analyzing non-polar and neutral compounds in biological samples. Next, a microfluidic-based liquid chromatography-electrospray ionization-mass spectrometric system (HPLC-Chip/ESI/MS) was compared to a conventional liquid chromatography-electrospray/mass spectrometric system (LC-ESI/MS) for the analysis of steroids. The sensitivity of the HPLC-Chip/MS system was ~50 times higher compared to the conventional LC-MS when injected amounts are compared. The feasibility of the HPLC-Chip/MS system in the analyses of non-derivatized steroids was also tested and the developed method was used in the analysis of mouse plasma samples. An ion mobility mass spectrometry method using a compact traveling wave cell (TWIM-MS) was also developed for the separation of steroid isomers. Three steroid isomer pairs were analyzed in their native form and as their p-toluenesulfonyl isocyanate derivatives. The results show, that the developed TWIM-MS method provides a reliable, fast and repeatable method for separating derivatized steroid isomers. Finally, an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI-MS/MS) method was developed for the simultaneous analysis of oxysterols and vitamin D related compounds in mouse brain and cell line samples. An UHPLC-APPI-high resolution mass spectrometric method was developed for confirmatory analysis and for the identification of non-targeted oxysterols. Several oxysterols were quantified in the mouse brain and cell line samples. Additionally, 25-hydroxyvitamin D3 was detected in mouse brain samples for the first time. In conclusion, the methods developed in this work are sensitive and selective new methods for the analysis of the selected lipids from biological samples. The methods provide new insights in the analysis of steroids, vitamin D related compounds and oxysterols, and with some additional development some of the presented methods could be implemented e.g. in routine laboratories.Steroidit, d-vitamiini sekä kolesterolin hapetustuotteet ovat kaikki osa yhdisteryhmää nimeltään lipidit. Lipidit ovat hyvin tärkeitä ihmiskehon toiminnassa ja monesti näiden yhdisteiden pitoisuuksien seuraaminen voi olla tärkeää esim. sairauksien diagnostiikassa. Tästä syystä herkkien ja selektiivisten menetelmien kehitys on tärkeää näiden yhdisteiden analytiikassa. Työn tarkoituksena oli kehittää ja testata uusia massa spektrometriaan pohjautuvia menetelmiä valittujen lipidien analytiikassa. Ensin kehitettiin mikroilmanpainefotoionisaatio-kapillaari nestekromatografia-tandem massaspektrometriaa hyödyntävä analyysimenetelmä anabolisten steroidien määritykseen virtsanäytteistä. Tulokset osoittavat, että tämä uutta tekniikkaa hyödyntävä menetelmä saattaa soveltua poolittomien ja neutraalien yhdisteiden analysointiin biologisista näytteistä. Seuraavaksi kehitettiin mikrofluidistiikkaan pohjautuva nestekromatografia-sähkösumutus-massaspektrometria menetelmä, jota verrattiin perinteiseen nestekromatografia-massaspektrometriseen menetelmään steroidien määrityksessä. Vertailun johtopäätös on, että mikrofluidistiikkaan pohjautuva menetelmä on parhaimmillaan n. 50 kertaa herkempi perinteiseen menetelmään verrattuna. Mikrofluidistiikkaan pohjautuvaa tekniikkaa kokeiltiin hiirten plasmanäytteiden analysointiin. Ioniliikkuvuutta hyödynnettiin tämän tutkimuksen puitteissa steroidien isomeerien erottamiseen toisistaan. Tähän tarkoitukseen steroideista tehtiin johdos ja tulokset osoittavat luotettavan, nopean ja toistettavan menetelmän steroidien isomeerien erotukseen. Tutkimuksen viimeisessä osassa kehitettiin kaksi korkean suorituskyvyn nestekromatografia-massaspektrometrista menetelmää kolesterolin hapetustuotteiden sekä d-vitamiinin analysointiin solulinja- ja hiirten aivonäytteistä. Ensimmäinen menetelmä oli tandem massaspektrometriaan pohjautuva menetelmä, jota käytettiin pitoisuusmäärityksiin. Toinen menetelmä puolestaan oli lentoaikamassaspektrometriaa hyödyntävä menetelmä, jota käytettiin tulosten vahvistukseen sekä tuntemattomien hapetustuotteiden tunnistukseen. Kehitettyjen menetelmien avulla kyettiin määrittämään monen kolesterolin hapetustuotteen pitoisuudet näytteissä. Lisäksi havaittiin yksi d-vitamiinin metaboliatuotteista hiirten aivonäytteistä. Tässä työssä kehitetyt menetelmät ovat herkkiä ja selektiivisiä uusia menetelmiä valittujen lipidien analysointiin biologisista näytteistä. Kehitetyt menetelmät tuovat uusia näkökulmia steroidien, d-vitamiinin ja kolesterolin hapetustuotteiden analysointiin ja pienellä jatkokehityksellä näitä menetelmiä voisi myös hyödyntää rutiinilaboratorioissa

The Extension of Surgery Predicts Acute Postoperative Pain, While Persistent Postoperative Pain Is Related to the Spinal Pathology in Adolescents Undergoing Posterior Spinal Fusion

Persistent pain after posterior spinal fusion affects 12 to 42% of patients with adolescent idiopathic scoliosis. The incidence of persistent pain among surgically treated children with Scheuermann kyphosis and spondylolisthesis is not known. The aim of our study was to determine the predictors and incidence of acute and chronic postoperative pain in adolescents undergoing posterior spinal fusion surgery. The study was a retrospective analysis of a prospectively collected pediatric spine register data. The study included 213 consecutive patients (158 AIS, 19 Scheuermann kyphosis, and 36 spondylolisthesis), aged 10–21 years undergoing posterior spinal fusion at a university hospital between March 2010 and March 2020. The mean (SD) daily postoperative opioid consumption per kilogram was significantly lower in the spondylolisthesis patients 0.36 mg/kg/day (0.17) compared to adolescent idiopathic scoliosis 0.51 mg/kg/day (0.25), and Scheuermann kyphosis 0.52 mg/kg/day (0.25) patients after surgery (p = 0.0004). Number of levels fused correlated with the daily opioid consumption (rs = 0.20, p = 0.0082). The SRS-24 pain domain scores showed a statistically significant improvement from preoperative levels to two-year follow-up in all three groups (p ≤ 0.03 for all comparisons). The spondylolisthesis patients had the lowest SRS pain domain scores (mean 4.04, SD 0.94), reporting more pain two years after surgery, in comparison to AIS (mean 4.31, SD 0.60) (p = 0.043) and SK (mean 4.43, SD 0.48) patients (p = 0.049). Persistent postoperative pain in adolescents undergoing posterior spinal fusion is related to disease pathology while higher acute postoperative pain is associated with a more extensive surgery. Spondylolisthesis patients report more chronic pain after surgery compared to AIS and SK patients

Beam-Like rods do not Provide Additional Improvement to Thoracic Kyphosis Restoration when Compared to Sagittal Reinforced rods in Adolescents Undergoing Spinal Fusion with Pedicle Screw Instrumentation for Idiopathic Scoliosis

Publisher Copyright: © 2022 The AuthorsObjective: Operative treatment of adolescent idiopathic scoliosis (AIS) with posterior spinal fusion aims for three-dimensional correction of coronal curve and thoracic kyphosis. Our aim was to compare two different designs of asymmetrical rods in adolescents who underwent a posterior spinal fusion with pedicle screw instrumentation for AIS with an emphasis on thoracic kyphosis restoration. Methods: This study was made with 76 consecutive adolescents (mean age 15.6 years, SD 2.0). Thirty-nine patients were operated with sagittal reinforced rods and 37 patients were operated with beam-like rods. The clinical and radiological results were assessed preoperatively, postoperatively, and during the follow-up visits at the outpatient clinic 6 months and 2 years after the surgery. Results: At the last follow-up, the mean (SD) major thoracic curves were 13° (6.2°) and 13° (6.0°) (P = 0.717). Correction percentages were 75% in the sagittal reinforced group and 73% in the beam-like rod group (P = 0.517). The mean (SD) thoracic kyphosis was 24° (11°) and 22° (7.8°) at the two year follow-up in the sagittal reinforced rod group and beamlike rod group (P = 0.517). There was a slight negative correlation between the major curve correction and thoracic kyphosis change in both groups, although this was not statistically significant (R = −0.19, P = 0.094 in the sagittal reinforced rod group, R=−0.16, P = 0.180 in the beam like rod group). Conclusions: There are no significant differences in the coronal or sagittal deformity restoration in adolescent patients who underwent a posterior spinal fusion with sagittal reinforced rods and beam-like rods for adolescent idiopathic scoliosis.Peer reviewe

Lipidomics in biomedical research-practical considerations

Lipids have many central physiological roles including as structural components of cell membranes, energy storage sources and intermediates in signaling pathways. Lipid-related disturbances are known to underlie many diseases and their co-morbidities. The emergence of lipidomics has empowered researchers to study lipid metabolism at the cellular as well as physiological levels at a greater depth than was previously possible. The key challenges ahead in the field of lipidomics in medical research lie in the development of experimental protocols and in silico techniques needed to study lipidomes at the systems level. Clinical questions where lipidomics may have an impact in healthcare settings also need to be identified, both from the health outcomes and health economics perspectives. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.Peer reviewe

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

Serum, plasma and erythrocyte membrane lipidomes in infants fed formula supplemented with bovine milk fat globule membranes

BACKGROUND: Supplementation of formula with bovine milk fat globule membranes has been shown to narrow the gap in immunological and cognitive development between breast-fed and formula-fed infants. METHOD: In a double-blinded randomized controlled trial 160 formula-fed infants received an experimental formula (EF), supplemented with bovine milk fat globule membranes, or standard formula until 6 months of age. A breast-fed reference group was recruited. Lipidomic analyses were performed on plasma and erythrocyte membranes at 6 months and on serum at 4 and 12 months of age. RESULTS: At 6 months of age, we observed a significant separation in the plasma lipidome between the two formula groups, mostly due to differences in concentrations of sphingomyelins (SM), phosphatidylcholines (PC), and ceramides, and in the erythrocyte membrane lipidome, mostly due to SMs, PEs and PCs. Already at 4 months, a separation in the serum lipidome was evident where SMs and PCs contributed. The separation was not detected at 12 months. CONCLUSIONS: The effect of MFGM supplementation on the lipidome is likely part of the mechanisms behind the positive cognitive and immunological effects of feeding the EF previously reported in the same study population.Peer reviewe

A longitudinal plasma lipidomics dataset from children who developed islet autoimmunity and type 1 diabetes

Early prediction and prevention of type 1 diabetes (T1D) are currently unmet medical needs. Previous metabolomics studies suggest that children who develop T1D are characterised by a distinct metabolic profile already detectable during infancy, prior to the onset of islet autoimmunity. However, the specificity of persistent metabolic disturbances in relation T1D development has not yet been established. Here, we report a longitudinal plasma lipidomics dataset from (1) 40 children who progressed to T1D during follow-up, (2) 40 children who developed single islet autoantibody but did not develop T1D and (3) 40 matched controls (6 time points: 3, 6, 12, 18, 24 and 36 months of age). This dataset may help other researchers in studying age-dependent progression of islet autoimmunity and T1D as well as of the age-dependence of lipidomic profiles in general. Alternatively, this dataset could more broadly used for the development of methods for the analysis of longitudinal multivariate data.Peer reviewe

Cord-Blood Lipidome in Progression to Islet Autoimmunity and Type 1 Diabetes

Previous studies suggest that children who progress to type 1 diabetes (T1D) later in life already have an altered serum lipid molecular profile at birth. Here, we compared cord blood lipidome across the three study groups: children who progressed to T1D (PT1D; n = 30), children who developed at least one islet autoantibody but did not progress to T1D during the follow-up (P1Ab; n = 33), and their age-matched controls (CTR; n = 38). We found that phospholipids, specifically sphingomyelins, were lower in T1D progressors when compared to P1Ab and the CTR. Cholesterol esters remained higher in PT1D when compared to other groups. A signature comprising five lipids was predictive of the risk of progression to T1D, with an area under the receiver operating characteristic curve (AUROC) of 0.83. Our findings provide further evidence that the lipidomic profiles of newborn infants who progress to T1D later in life are different from lipidomic profiles in P1Ab and CTR

Cord-Blood Lipidome in Progression to Islet Autoimmunity and Type 1 Diabetes

Previous studies suggest that children who progress to type 1 diabetes (T1D) later in life already have an altered serum lipid molecular profile at birth. Here, we compared cord blood lipidome across the three study groups: children who progressed to T1D (PT1D; n = 30), children who developed at least one islet autoantibody but did not progress to T1D during the follow-up (P1Ab; n = 33), and their age-matched controls (CTR; n = 38). We found that phospholipids, specifically sphingomyelins, were lower in T1D progressors when compared to P1Ab and the CTR. Cholesterol esters remained higher in PT1D when compared to other groups. A signature comprising five lipids was predictive of the risk of progression to T1D, with an area under the receiver operating characteristic curve (AUROC) of 0.83. Our findings provide further evidence that the lipidomic profiles of newborn infants who progress to T1D later in life are different from lipidomic profiles in P1Ab and CTR