•Quantitative steroid analysis via online SPE LC–MS/MS within 4min total run time.•Uniform procedure increases convenience in sample preparation regardless the matrix.•In complex matrices ...quantitation by MS3 proves to be superior to MS2.•Hair cortisol analysis suffers from massive pre-analytical issues, often unconsidered.
Steroid analysis is being conquered by liquid chromatography tandem mass spectrometry (LC–MS/MS) benefiting from higher standardization, selectivity and diversity. Regarding high throughput in routine diagnostics rapid chromatography is mandatory. Introducing MS3 (MS/MS/MS), specificity of mass spectrometric detection can be enhanced without sacrificing analysis time. 100μL of human plasma/serum, saliva, urine and 10–20mg of hair are used for the simultaneous quantification of 17α-hydroxyprogesterone, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC–MS/MS or LC–MS3. Steroids can be analyzed in 4min after a single manual dilution and protein precipitation step. In complex sample matrices like hair MS3 detection was found to be appropriate for quantitation. Lower limits of quantitation ranged from 37pmol/L (estradiol) up to 3.1nmol/L (DHEAS). General accuracy was 89–107% with between-run imprecision ≤10%. Comparison to immunoassays revealed significant differences in quantitation for urinary cortisol (−71% mean), aldosterone (−40% mean) and plasma aldosterone (−45% mean). The comparison of MS2 and MS3 quantitation of hair cortisol also revealed significant differences. In general, quantitation via MS3 was not applicable for a long time. But with the current generation of mass spectrometers quantitation via MS3 can be superior to MS2 regarding specificity and accuracy when dealing with matrix issues. However, drawbacks regarding flexibility and precision have to be taken into account. Concludingly, simple protein precipitation combined with rapid online SPE LC–MS/MS/MS allows us to quantify over broad, essential concentration ranges in human serum, saliva, urine and hair.
Abstract
Aims
Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development ...and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism.
Methods and results
Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E−/− (Apoe−/−) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe−/− mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL −15.9 mg/dL (−8.1%) vs. −1.6 mg/dL (−0.5%), P = 0.016, total −19.6 mg/dL (−7.3%) vs. −5.3 mg/dL (−1.7%), P = 0.014 and non-high-density lipoprotein cholesterol levels PA vs. placebo: −18.9 mg/dL (−9.1%) vs. −0.6 mg/dL (−0.5%), P = 0.002 in subjects with elevated baseline LDL cholesterol levels.
Conclusion
Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.
Graphical Abstract
Graphical Abstract
The figure illustrates the proposed model of the cholesterol-lowering and atheroprotective properties of propionate. A high-fat high-cholesterol diet causes a disbalance of immune cells in the small intestinal microenvironment, with reduced regulatory T cell frequencies and interleukin-10 concentrations. Altered regulatory T cell levels are rescued upon exogenous propionate supplementation, with increased local levels. This in turn modulates NPC1L1 expression and membrane density, with a subsequent reduction in cholesterol absorption, ultimately leading to reduced atherogenesis. The illustration was adopted from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License. HFD, high-fat diet; IL-10, interleukin-10; NPC1L1, Niemann-Pick C1-like 1; PA, propionic acid; Treg, regulatory T cell.
The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or ...transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids (HCAR) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA1, HCA2) but humans and other hominids contain a third member (HCA3) in their genomes. A plausible hypothesis why HCA3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA3 in vitro and in vivo. The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA3. D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA3-dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.
Obesity arising from excessive dietary fat intake is a risk factor for cognitive decline, dementia and neurodegenerative diseases, including Alzheimer’s disease. Here, we studied the effect of ...long-term high-fat diet (HFD) (24 weeks) and return to normal diet (ND) on behavioral features, microglia and neurons in adult male C57BL/6J mice. Consequences of HFD-induced obesity and dietary changes on general health (coat appearance, presence of vibrissae), sensory and motor reflexes, learning and memory were assessed by applying a phenotypic assessment protocol, the Y maze and Morris Water Maze test. Neurons and microglia were histologically analyzed within the mediobasal hypothalamus, hippocampus and frontal motor cortex after long-term HFD and change of diet. Long periods of HFD caused general health issues (coat alterations, loss of vibrissae), but did not affect sensory and motor reflexes, emotional state, memory and learning. Long-term HFD increased the microglial response (increased Iba1 fluorescence intensity, percentage of Iba1-stained area and Iba1 gene expression) within the hypothalamus, but not in the cortex and hippocampus. In neither of these regions, neurodegeneration or intracellular lipid droplet accumulation was observed. The former alterations were reversible in mice whose diet was changed from HFD to ND. Taken together, long periods of excessive dietary fat alone do not cause learning deficits or spatial memory impairment, though HFD-induced obesity may have detrimental consequences for cognitive flexibility. Our data confirm the selective responsiveness of hypothalamic microglia to HFD.
•Overview of the LC–MS/MS methods for the analysis of eicosanoids published in the last decade.•Presentation of analytical details of 40 published methods.•Summary of investigated diseases is ...presented.•Discussion of preanalytical and methodical aspects of eicosanoid analysis.
Today, there is an increasing number of liquid chromatography tandem-mass spectrometric (LC–MS/MS) methods for the analysis of eicosanoids and related lipids in biological matrices. An overview of currently applied LC–MS/MS methods is given with attention to sample preparation strategies, chromatographic separation including ultra high performance liquid chromatography (UHPLC) and chiral separation, as well as to mass spectrometric detection using multiple reacting monitoring (MRM). Further, the application in recent clinical research is reviewed with focus on preanalytical aspects prior to LC–MS/MS analysis as well as applications in major diseases of Western civilization including respiratory diseases, diabetes, cancer, liver diseases, atherosclerosis, and neurovascular diseases.
•Reference intervals of steroid hormones from 0.3 to 80 years old for LC–MS/MS.•Continuous age-dependent reference intervals of steroid hormones with GAMLSS.•Androgen and estrogen surge below the age ...of 1 showed mini-puberty.•High aldosterone- and cortisone-levels and low cortisol levels below the age of 1.•Effect of oral contraceptives on the increase of cortisol levels.
New reference intervals need to be established for a new analytical method with improved sensitivity and specificity. We aimed to establish the new reference intervals from infancy to senescence of nine steroid hormones (cortisol, cortisone, progesterone, 17-hydroxyprogesterone (17−OHP), androstenedione, testosterone, estradiol, DHEAS, and aldosterone) for LC–MS/MS method.
Serum samples from 4678 reference individuals (age range: 0.3–79 years) were measured with LC–MS/MS. Samples were collected between 7 a.m. and 10 a.m. Exclusion criteria were concomitant endocrine diseases and body mass index ≥ 33. Generalized additive model for location, scale and shape, the nonparametric or robust method was applied.
We established the reference intervals of the nine steroid hormones by sex, age, and pubertal stage. Below the age of one, we observed the surge of androgen and estrogen which implied mini-puberty. At the same period of life, aldosterone and cortisone levels were very high reflecting physiological hyperaldosteronism. An increase of steroid hormones during the pubertal development and slow decrease towards senescence after the peak at early adulthood were observed. Due to the increase of CBG synthesis, cortisol levels were increased under oral contraceptives (OC) significantly (p < 0.0001), while OC suppressed progesterone, 17−OHP, androstenedione, and estradiol (p < 0.0001).
Our results will facilitate the interpretation of patient data in routine diagnostics with the use of LC–MS/MS method. Since LC–MS/MS methods have shown good comparability among the different laboratories, our reference intervals can be further adopted in other laboratories equipped with LC–MS/MS, once the validation with a small number of reference samples is performed.
Phytosterol serum concentrations are under tight genetic control. The relationship between phytosterols and coronary artery disease (CAD) is controversially discussed. We perform a genome-wide ...meta-analysis of 32 phytosterol traits reflecting resorption, cholesterol synthesis and esterification in six studies with up to 9758 subjects and detect ten independent genome-wide significant SNPs at seven genomic loci. We confirm previously established associations at ABCG5/8 and ABO and demonstrate an extended locus heterogeneity at ABCG5/8 with different functional mechanisms. New loci comprise HMGCR, NPC1L1, PNLIPRP2, SCARB1 and APOE. Based on these results, we perform Mendelian Randomization analyses (MR) revealing a risk-increasing causal relationship of sitosterol serum concentrations and CAD, which is partly mediated by cholesterol. Here we report that phytosterols are polygenic traits. MR add evidence of both, direct and indirect causal effects of sitosterol on CAD.
Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the ...late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.
Abstract
Context
Steroid hormones are important regulators of physiological processes in humans and are under genetic control. A link to coronary artery disease (CAD) is supposed.
Objective
Our main ...objective was to identify genetic loci influencing steroid hormone levels. As a secondary aim, we searched for causal effects of steroid hormones on CAD.
Design
We conducted genome-wide meta-association studies for eight steroid hormones: cortisol, dehydroepiandrosterone sulfate (DHEAS), estradiol, and testosterone in two independent cohorts (LIFE-Adult, LIFE-Heart, maximum n = 7667), and progesterone, 17-hydroxyprogesterone, androstenedione, and aldosterone in LIFE-Heart only (maximum n = 2070). All genome-wide significant loci were tested for sex interactions. Furthermore, we tested whether previously reported CAD single-nucleotide polymorphisms were associated with our steroid hormone panel and investigated causal links between hormone levels and CAD status using Mendelian randomization (MR) approaches.
Results
We discovered 15 novel associated loci for 17-hydroxyprogesterone, progesterone, DHEAS, cortisol, androstenedione, and estradiol. Five of these loci relate to genes directly involved in steroid metabolism, that is, CYP21A1, CYP11B1, CYP17A1, STS, and HSD17B12, almost completing the set of steroidogenic enzymes with genetic associations. Sexual dimorphisms were found for seven of the novel loci. Other loci correspond, for example, to the WNT4/β-catenin pathway. MR revealed that cortisol, androstenedione, 17-hydroxyprogesterone, and DHEA-S had causal effects on CAD. We also observed enrichment of cortisol and testosterone associations among known CAD hits.
Conclusion
Our study greatly improves insight into genetic regulation of steroid hormones and their dependency on sex. These results could serve as a basis for analyzing sexual dimorphism in other complex diseases.
Genome-wide analysis of eight steroid hormones revealed 15 novel loci involving several steroid-converting enzymes. Mendelian randomization found four steroids causally related to coronary artery disease.
Apolipoproteins are major structural and functional constituents of lipoprotein particles. As modulators of lipid metabolism, adipose tissue biology, and energy homeostasis, apolipoproteins may serve ...as biomarkers or potential therapeutic targets for cardiometabolic diseases. Mice are the preferred model to study metabolic disease and CVD, but a comprehensive method to quantify circulating apolipoproteins in mice is lacking. We developed and validated a targeted proteomics assay to quantify eight apolipoproteins in mice via proteotypic signature peptides and corresponding stable isotope-labeled analogs. The LC/MS/MS method requires only a 3 µl sample volume to simultaneously determine mouse apoA-I, apoA-II, apoA-IV, apoB-100, total apoB, apoC-I, apoE, and apoJ concentrations. ApoB-48 concentrations can be calculated by subtracting apoB-100 from total apoB. After we established the analytic performance (sensitivity, linearity, and imprecision) and compared results for selected apolipoproteins against immunoassays, we applied the method to profile apolipoprotein levels in plasma and isolated HDL from normocholesterolemic C57BL/6 mice and from hypercholesterolemic Ldl-receptor- and Apoe-deficient mice. In conclusion, we present a robust, quantitative LC/MS/MS method for the multiplexed analysis of eight apolipoproteins in mice. This assay can be applied to investigate the effects of genetic manipulation or dietary interventions on apolipoprotein levels in plasma and isolated lipoprotein fractions.
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