Exploring the kynurenine pathway in mild traumatic brain injury: A longitudinal study.

A potential source of novel biomarkers for mTBI is the kynurenine pathway (KP), a metabolic pathway of tryptophan (Trp), that is up-regulated by neuroinflammation and stress. Considering that metabolites of the KP (kynurenines) are implicated in various neuropsychiatric diseases, exploration of this pathway could potentially bridge the gap between physiological and psychological factors in the recovery process after mTBI. This study, therefore, set out to characterize the KP after mTBI and to examine associations with long-term outcome. Patients were prospectively recruited at the emergency department (ED), and blood samples were obtained in the acute phase (<24 h; N = 256) and at 1-month follow-up (N = 146). A comparison group of healthy controls (HC; N = 32) was studied at both timepoints. Trp, kynurenines, and interleukin (IL)-6 and IL-10 were quantified in plasma. Clinical outcome was measured at six months post-injury. Trp, xanthurenic acid (XA), and picolinic acid (PA) were significantly reduced in patients with mTBI relative to HC, corrected for age and sex. For Trp (d = -0.57 vs. d = -0.29) and XA (d = -0.98 vs. d = -0.32), larger effects sizes were observed during the acute phase compared to one-month follow-up, while for PA (d = -0.49 vs. d = -0.52) effect sizes remained consistent. Findings for other kynurenines (e.g., kynurenine, kynurenic acid, and quinolinic acid) were non-significant after correction for multiple testing. Within the mTBI group, lower acute Trp levels were significantly related to incomplete functional recovery and higher depression scores at 6 months post-injury. No significant relationships were found for Trp, XA, and PA with IL-6 or IL-10 concentrations. In conclusion, our findings indicate that perturbations of the plasma KP in the hyperacute phase of mTBI and 1 month later are limited to the precursor Trp, and glutamate system modulating kynurenines XA and PA. Correlations between acute reductions of Trp and unfavorable outcomes may suggest a potential substrate for pharmacological intervention.

Simultaneous mass spectrometric quantification of trace amines, their precursors and metabolites.

OBJECTIVES: Trace amines are powerful neuromodulators influencing the release and reuptake of catecholamines. These low concentrated endogenous amines impact mood, cognition, and hormone regulation. Dysregulation of trace amines have been associated with a variety of diseases, such as schizophrenia, Parkinson's disease, migraine, depression and more. Succesfull simultaneous quantification of trace amines, their precursors and metabolites would benefit both research and patient care. Since these compounds have various functional groups and are present in biological matrices with large concentration difference, their simultaneous quantification is an analytical challenge. Our goal was to develop a highly sensitive LC-MS/MS assay to simultaneously quantify trace amines, their precursors and metabolites in plasma. METHODS: Our method is based on a simple two-step in-matrix derivatization protocol: propionic anhydride (PA) and 3-Ethyl-1-[3-(dimethylamino)propyl]carbodiimide (EDC) in combination with 2,2,2-trifluoroethylamine (TFEA) followed by online solid phase extraction combined with LC-MS/MS. Fifteen metabolites can be measured simultaneously, three precursors, eight trace amines and four metabolites. Validation of this method was performed according to international validation guidelines. The pre-analytical stability of trace amines was assessed. RESULTS: This novel method was successful in quantifying trace amines, their precursors, and metabolites in plasma. Using just 50 µl human plasma, we were able to accomplish limit of quantification for 2-phenylethylamine and N-methyl-phenylethylamine of 0.2 nmol/L and 0.1 nmol/L for tyramine and n-methyltyramine. Inter-and intra-assay imprecision was < 15 % for all analytes. Stability assessment showed susceptibility of certain trace amines e.g. 2-phenylethylamine and N-methyl-phenylethylamine to enzymatic degradation in plasma. The addition of the monoamine oxidase inhibitor pargyline to plasma prevented this enzymatic degradation. CONCLUSIONS: We developed a novel LC-MS/MS method that1) uses a new double derivatization technique, 2) is automated with online SPE, 3) uses far less sample volume then previous methods and 4) detects more components in the same sample (eight trace amines, three precursors, and four metabolites) with high specificity and selectivity. Furthermore, addition of MAO A/B inhibitor prevents degradation and guarantees more accurate quantification of trace amines.

Association of Fetal Catecholamines With Neonatal Hypoglycemia.

Importance: Perinatal stress and fetal growth restriction increase the risk of neonatal hypoglycemia. The underlying pathomechanism is poorly understood. In a sheep model, elevated catecholamine concentrations were found to suppress intrauterine insulin secretion, followed by hyperresponsive insulin secretion once the adrenergic stimulus subsided. Objective: To determine whether neonates with risk factors for hypoglycemia have higher catecholamine concentrations in umbilical cord blood (UCB) and/or amniotic fluid (AF) and whether catecholamines are correlated with postnatal glycemia. Design, Setting, and Participants: In a prospective cohort study of 328 neonates at a tertiary perinatal center from September 2020 through May 2022 in which AF and UCB were collected immediately during and after delivery, catecholamines and metanephrines were analyzed using liquid chromatography with tandem mass spectrometry. Participants received postnatal blood glucose (BG) screenings. Exposure: Risk factor for neonatal hypoglycemia. Main Outcomes and Measures: Comparison of catecholamine and metanephrine concentrations between at-risk neonates and control participants, and correlation of concentrations of catecholamines and metanephrines with the number and severity of postnatal hypoglycemic episodes. Results: In this study of 328 neonates (234 in the risk group: median [IQR] gestational age, 270 [261-277] days; and 94 in the control group: median [IQR] gestational age, 273 [270-278] days), growth-restricted neonates showed increased UCB median (IQR) concentrations of norepinephrine (21.10 [9.15-42.33] vs 10.88 [5.78-18.03] nmol/L; P < .001), metanephrine (0.37 [0.13-1.36] vs 0.12 [0.08-0.28] nmol/L; P < .001), and 3-methoxytyramine (0.149 [0.098-0.208] vs 0.091 [0.063-0.149] nmol/L; P = .001). Neonates with perinatal stress had increased UCB median (IQR) concentrations of norepinephrine (22.55 [8.99-131.66] vs 10.88 [5.78-18.03] nmol/L; P = .001), normetanephrine (1.75 [1.16-4.93] vs 1.25 [0.86-2.56] nmol/L; P = .004), and 3-methoxytyramine (0.120 [0.085-0.228] vs 0.091 [0.063-0.149] nmol/L; P = .008) (P < .0083 was considered statistically significant). Concentrations of UCB norepinephrine, metanephrine, and 3-methoxytyramine were negatively correlated with AF C-peptide concentration (rs = -0.212, P = .005; rs = -0.182, P = .016; and rs = -0.183, P = .016, respectively [P < .017 was considered statistically significant]). Concentrations of UCB norepinephrine, metanephrine, and 3-methoxytyramine were positively correlated with the number of hypoglycemic episodes (BG concentration of 30-45 mg/dL) (rs = 0.146, P = .01; rs = 0.151, P = .009; and rs = 0.180, P = .002, respectively). Concentrations of UCB metanephrine and 3-methoxytyramine were negatively correlated with the lowest measured BG concentration (rs = -0.149, P = .01; and rs = -0.153, P = .008, respectively). Conclusions and Relevance: Neonates at risk for hypoglycemia displayed increased catecholamine and metanephrine concentrations that were correlated with postnatal hypoglycemic episodes and lower BG levels; these results are consistent with findings in a sheep model that fetal catecholamines are associated with neonatal ß-cell physiology and that perinatal stress or growth restriction is associated with subsequent neonatal hyperinsulinemic hypoglycemia. Improving the pathomechanistic understanding of neonatal hypoglycemia may help to guide management of newborns at risk for hypoglycemia.

Effects of endocrine disrupting chemicals and their interactions with genetic risk scores on cardiometabolic traits.

Ubiquitous non-persistent endocrine disrupting chemicals (EDCs) have inconsistent associations with cardiometabolic traits. Additionally, large-scale genome-wide association studies (GWASs) have yielded many genetic risk variants for cardiometabolic traits and diseases. This study aimed to investigate the associations between a wide range of EDC exposures (parabens, bisphenols, and phthalates) and 14 cardiometabolic traits and whether these are moderated by their respective genetic risk scores (GRSs). Data were from 1074 participants aged 18 years or older of the Lifelines Cohort Study, a large population-based biobank. GRSs for 14 cardiometabolic traits were calculated based on genome-wide significant common variants from recent GWASs. The concentrations of 15 EDCs in 24-hour urine were measured by isotope dilution liquid chromatography tandem mass spectrometry technology. The main effects of trait-specific GRSs and each of the EDC exposures and their interaction effects on the 14 cardiometabolic traits were examined in multiple linear regression. The present study confirmed significant main effects for all GRSs on their corresponding cardiometabolic trait. Regarding the main effects of EDC exposures, 26 out of 280 EDC-trait tests were significant with explained variances ranging from 0.43 % (MMP- estimated glomerular filtration rate (eGFR)) to 2.37 % (PrP-waist-hip ratio adjusted body mass index (WHRadjBMI)). We confirmed the association of MiBP and MBzP with WHRadjBMI and body mass index (BMI), and showed that parabens, bisphenol F, and many other phthalate metabolites significantly contributed to the variance of WHRadjBMI, BMI, high-density lipoprotein (HDL), eGFR, fasting glucose (FG), and diastolic blood pressure (DBP). Only one association between BMI and bisphenol F was nominally significantly moderated by the GRS explaining 0.36 % of the variance. However, it did not survive multiple testing correction. We showed that non-persistent EDC exposures exerted effects on BMI, WHRadjBMI, HDL, eGFR, FG, and DBP. However no evidence for a modulating role of GRSs was found.

Discriminatory Value of Steroid Hormones on Polycystic Ovary Syndrome and Clustering of Hyperandrogenism and Metabolic Factors.

OBJECTIVE: We determined (1) if 11-oxygenated androgens better identify polycystic ovary syndrome (PCOS) diagnosis in women with obesity compared to total or free testosterone (T) and free androgen index; (2) how biochemical hyperandrogenism and metabolic factors cluster in a cohort of women with infertility and obesity. METHODS: Women with obesity and PCOS comprised the study group (N = 132). Ovulatory women with obesity and idiopathic, tubal or male factor infertility were the control group (N = 83). Steroid hormones were measured by means of liquid chromatography tandem mass spectrometry. Receiver operating characteristic curves and principal component analysis were used. RESULTS: Women with obesity and PCOS had higher 11-ketotestosterone (11 KT) (1.22 nmol/L [0.84; 1.65] vs 1.05 [0.78; 1.35], P = .04) compared to controls, but not 11ß-hydroxyandrostenedione 4.30 [2.87; 5.92] vs 4.06 [3.22; 5.73], P = .44). 11-ketotestosterone (area under the curve: 0.59) did not better discriminate PCOS in women with obesity compared to: total T (0.84), free T (0.91), and free androgen index (0.85). We identified 4 principal components (PCs) in the PCOS group (72.1% explained variance): (1) insulin resistance status; (2) blood pressure; (3) obesity; (4) androgen status and 4 PCs in the control group (68.7% explained variance) with variables representing metabolism being dispersed in component 2, 3, and 4. CONCLUSIONS: Eleven-oxygenated androgens do not aid in the diagnosis of PCOS in women with obesity. Insulin resistance is the strongest PC in the PCOS group. There is no major dominant characteristic that defines obese non-PCOS women.

Simultaneous quantification of the 22-kDa isoforms of human growth hormone 1 and 2 in human plasma by multiplexed immunocapture and LC-MS/MS.

An LC-MS/MS method is presented for the simultaneous quantification of two structurally closely related protein biomarker isoforms, the 22-kDa isoforms of human growth hormone 1 and human growth hormone 2, in human plasma. It is based on multiplexed immunocapture using two monoclonal antibodies immobilized on magnetic beads, tryptic digestion and quantification of two specific signature peptides plus an additional peptide for estimation of total growth hormone related concentrations. A full validation according to international guidelines was performed across the clinically relevant concentration ranges of 0.5 to 50 ng/mL for growth hormone 1, and 2 to 50 ng/mL for growth hormone 2 and demonstrated satisfactory method performance in terms of accuracy, precision, stability and absence of interference. The method's applicability for routine analysis and its ability to effectively distinguish between GH1 and GH2 was demonstrated by the analysis of plasma samples from pregnant individuals to study the changes in growth hormone levels during pregnancy.

A genome-wide association study of 24-hour urinary excretion of endocrine disrupting chemicals.

Ubiquitous exposure to environmental endocrine disrupting chemicals (EDCs) instigates a major public health problem, but much remains unknown on the inter-individual differences in metabolism and excretion of EDCs. To examine this we performed a two-stage genome-wide association study (GWAS) for 24-hour urinary excretions of four parabens, two bisphenols, and nine phthalate metabolites. Results showed five genome-wide significant (p-value < 5x10-8) and replicated single nucleotide polymorphisms (SNPs) representing four independent signals that associated with mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP). Three of the four signals were located on chromosome 10 in a locus harboring the cytochrome P450 (CYP) genes CYP2C9, CYP2C58P, and CYP2C19 (rs117529685, pMECPP = 5.38x10-25; rs117033379, pMECPP = 1.96x10-19; rs4918798, pMECPP = 4.01x10-71; rs7895726, pMEHHP = 1.37x10-15, r2 with rs4918798 = 0.93). The other signal was on chromosome 6 close to the solute carrier (SLC) genes SLC17A1, SLC17A3, SLC17A4, and SCGN (rs1359232, pMECPP = 7.6x10-16). These four SNPs explained a substantial part (8.3 % - 9.2 %) of the variance in MECPP in the replication cohort. Bioinformatics analyses supported a likely causal role of CYP2C9 and SLC17A1 in metabolism and excretion of MECPP and MEHHP. Our results provide biological insights into mechanisms of phthalate metabolism and excretion with a likely causal role for CYP2C9 and SLC17A1.

Dynamics of maternal androgens and its metabolites during early embryonic development: embryonic modification of a maternal effect.

In birds, exposure to maternal (yolk) testosterone affects a diversity of offspring post-hatching traits, which eventually affect offspring competitiveness. However, maternal testosterone is heavily metabolized at very early embryonic developmental stages to hydrophilic metabolites that are often assumed to be much less biologically potent. Either the rapid metabolism could either keep the maternal testosterone from reaching the embryos, opening the possibility for a mother-offspring conflict or the metabolites may facilitate the uptake of the lipophilic testosterone from the yolk into the embryonic circulation after which they are either converted back to the testosterone or functioning directly as metabolites. To test these possibilities, we injected isotope-labeled testosterone (T-[D5]) into the yolk of freshly laid Rock pigeon (Columba livia) eggs and determined the concentration and distribution of T-[D5] and its labeled metabolites within different egg fractions by liquid chromatography combined with tandem mass spectrometry at day 2, 5 and 10 of incubation. Although under a supraphysiological dosage injection, yolk testosterone decreased within 2 days and was metabolized into androstenedione, conjugated testosterone, etiocholanolone and other components that were unidentifiable due to methodological limitation. We show for the first time that testosterone, androstenedione and conjugated testosterone, but not etiocholanolone, reached the embryo including its brain. Their high concentrations in the yolk and extraembryonic membranes suggest that conversion takes place here. We also found no sex-specific metabolism, explaining why maternal testosterone does not affect sexual differentiation. Our findings showed that maternal testosterone is quickly converted by the embryo, with several but not all metabolites reaching the embryo providing evidence for both hypotheses.

Plasticity in metabolism of maternal androgens in avian embryos.

Mothers can influence offspring phenotypes by transferring non-genetic information to the young, which provides them with a flexible tool to adjust the developmental trajectory of the young in fluctuating environments. Mothers can differentially deposit their resources in the same reproductive attempt in relation to the offspring position in the sibling hierarchy. However, whether embryos from different positions can be plastic in their response to the maternal signals, potentially leading to a mother-offspring conflict, is yet unclear. We used Rock pigeons (Columba livia), that lay two egg clutches where maternal androgen levels in second laid eggs at oviposition are higher than in first laid eggs, and investigated the plasticity of embryonic metabolism of maternal androgens. We experimentally elevated androstenedione and testosterone levels in first eggs to that present in second eggs and measured the change in androgen levels and its main metabolites (etiocholanolone and conjugated testosterone) after 3.5 days of incubation. We found that eggs with increased androgens show a different degree of androgen metabolism depending either on the egg laying sequence or initial androgen levels or both. Our findings indicate that embryos have certain plasticity in response to maternal androgen levels depending on maternal signals.

The circadian clock remains intact, but with dampened hormonal output in heart failure.

BACKGROUND: Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants. METHODS: We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4 h during a 24-h period in a light/darkness synchronised environment. FINDINGS: Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P = 0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P = 0.017) with a difference of 56.8 (95% CI 10.3-103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P = 0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients. Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations. INTERPRETATION: Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches. FUNDING: Hartstichting.

Brain Kynurenine Pathway Metabolite Levels May Reflect Extent of Neuroinflammation in ALS, FTD and Early Onset AD.

OBJECTIVES: Despite distinct clinical profiles, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients share a remarkable portion of pathological features, with a substantial percentage of patients displaying a mixed disease phenotype. Kynurenine metabolism seems to play a role in dementia-associated neuroinflammation and has been linked to both diseases. We aimed to explore dissimilarities in kynurenine pathway metabolites in these early onset neurodegenerative disorders in a brain-region-specific manner. METHODS: Using liquid chromatography mass spectrometry (LC-MS/MS), kynurenine metabolite levels were determined in the brain samples of 98 healthy control subjects (n = 20) and patients with early onset Alzheimer's disease (EOAD) (n = 23), ALS (n = 20), FTD (n = 24) or a mixed FTD-ALS (n = 11) disease profile. RESULTS: Overall, the kynurenine pathway metabolite levels were significantly lower in patients with ALS compared to FTD, EOAD and control subjects in the frontal cortex, substantia nigra, hippocampus and neostriatum. Anthranilic acid levels and kynurenine-to-tryptophan ratios were consistently lower in all investigated brain regions in ALS compared to the other diagnostic groups. CONCLUSIONS: These results suggest that the contribution of kynurenine metabolism in neuroinflammation is lower in ALS than in FTD or EOAD and may also be traced back to differences in the age of onset between these disorders. Further research is necessary to confirm the potential of the kynurenine system as a therapeutic target in these early onset neurodegenerative disorders.

An antibody-free LC-MS/MS method for the quantification of sex hormone binding globulin in human serum and plasma.

OBJECTIVES: Sex hormone binding globulin (SHBG) is a hormone binding protein which plays an important role in regulating the transport and availability of biologically active androgens and estradiol to target cells and used to calculate free testosterone concentrations. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, featuring an albumin removal step followed by a tryptic digestion. After a reduction step with dithiothreitol and alkylation with iodoacetamide three signature peptides were used for the quantification of SHBG. RESULTS: The method enables the quantification of serum and plasma SHBG over the clinically relevant range of 200-20,000 ng/mL and was validated according to the most recent guidelines. The LC-MS/MS method correlates well with the Abbott Alinity immunoassay (R2>0.95), but the LC-MS/MS results are on average 16-17% lower than the immunoassay results, which is consistent for all three signature peptides. CONCLUSIONS: The LC-MS/MS method which includes an albumin depletion step allows quantification of SHBG in serum and plasma without an immunocapture step at clinically relevant SHBG levels, thus contributing to better lab-to-lab consistency of results.

Concentration gradients of monoamines, their precursors and metabolites in serial lumbar cerebrospinal fluid of neurologically healthy patients determined with a novel LC-MS/MS technique.

BACKGROUND: Potential biomarkers for neuropsychiatric disorders are cerebrospinal fluid (CSF) monoamines and their corresponding precursors and metabolites. During CSF sampling, CSF flows towards the lumbar sampling site from more cranial regions. To compare the results of studies in which different CSF volumes were acquired, it is important to know if ventricular-lumbar concentration gradients exist. This has only been addressed for a few biogenic amines, and almost exclusively in neurologically unwell patients due to the burden of a lumbar puncture (necessary to obtain CSF). The aim of our study was to determine if concentration gradients exist for routinely measured CSF constituents and biogenic amines in neurologically healthy patients. We applied a novel ultrasensitive liquid chromatography mass spectrometry (LC-MS/MS) method for the simultaneous quantification of multiple monoamines, precursors and metabolites in CSF and plasma. METHODS: CSF and blood samples were collected from twenty neurologically healthy patients undergoing spinal anaesthesia. Ten mL of lumbar CSF was collected in five consecutive two mL fractions. We determined leucocyte and erythrocyte counts, glucose, albumin and protein concentrations and quantified monoamines, precursors and metabolites on each of the fractions using LC-MS/MS. RESULTS: In twenty patients (60% male; median age: 46 years), dopamine, DOPAC, 3-MT, HVA, noradrenaline, normetanephrine and 5-HIAA concentrations increased from the first to the last CSF fraction (all p < 0.001). CSF adrenaline concentrations were below the detection limit, whereas serotonin measurements were regarded as unreliable. Albumin and total protein levels decreased significantly across CSF fractions. CONCLUSIONS: A ventricular-lumbar CSF concentration gradient existed for most of the investigated analytes. This is a novel finding for dopamine, noradrenaline, 3-MT and normetanephrine. These results contribute to the understanding of the neurobiology and underline the importance of standardized procedures for CSF handling to allow comparisons between studies.

11ß-HSD1 inhibition in men mitigates prednisolone-induced adverse effects in a proof-of-concept randomised double-blind placebo-controlled trial.

Glucocorticoids prescribed to limit inflammation, have significant adverse effects. As 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) regenerates active glucocorticoid, we investigated whether 11ß-HSD1 inhibition with AZD4017 could mitigate adverse glucocorticoid effects without compromising their anti-inflammatory actions. We conducted a proof-of-concept, randomized, double-blind, placebo-controlled study at Research Unit, Churchill Hospital, Oxford, UK (NCT03111810). 32 healthy male volunteers were randomized to AZD4017 or placebo, alongside prednisolone treatment. Although the primary endpoint of the study (change in glucose disposal during a two-step hyperinsulinemic, normoglycemic clamp) wasn't met, hepatic insulin sensitivity worsened in the placebo-treated but not in the AZD4017-treated group. Protective effects of AZD4017 on markers of lipid metabolism and bone turnover were observed. Night-time blood pressure was higher in the placebo-treated but not in the AZD4017-treated group. Urinary (5aTHF+THF)/THE ratio was lower in the AZD4017-treated but remained the same in the placebo-treated group. Most anti-inflammatory actions of prednisolone persisted with AZD4017 co-treatment. Four adverse events were reported with AZD4017 and no serious adverse events. Here we show that co-administration of AZD4017 with prednisolone in men is a potential strategy to limit adverse glucocorticoid effects.

Urinary concentrations of bisphenols and parabens and their association with attention, hyperactivity and impulsivity at adolescence.

BACKGROUND: Neurobehavioural disorder diagnoses have been increasing over the last decades, leading to heightened interest in the aetiological factors involved. Endocrine disrupting chemicals, such as parabens and bisphenols, have been suggested as one of those factors. It is unknown whether exposure during adolescence may affect neurobehavioural development. OBJECTIVE: To determine whether urinary concentrations of parabens and bisphenols are associated with attention and concentration in adolescents, in general and sex-specific. METHODS: We invited 188 adolescents (13-15 years old) for the follow-up birth cohort-study. Concentrations of five parabens and three bisphenols (BPA; BPF; BPS) were measured in morning urine after overnight fasting, using a validated LC-MS/MS method. Attention and concentration were assessed at the clinic with subtests of the Test of Everyday Attention in Children and the Dutch Attention Deficit Hyperactivity Disorder questionnaire (AVL), the latter being filled in by parents. Linear regression analyses were performed, adjusting for urine creatinine concentrations and potential confounding factors. RESULTS: 101 (54%) adolescents participated (46 girls; 55 boys). Urinary paraben concentrations were higher in girls than in boys. Methylparaben was positively associated with attention in girls (p ≤ .05; B= -2.836; 95%CI= -5.175;-.497), ethylparaben negatively with hyperactivity (p ≤ .05; B= -1.864; 95%CI= -3.587;-.141). Butylparaben was associated with more optimal scores on parent reported attention. Propylparaben was negatively associated with scores on sustained auditory attention in girls (p ≤ .10; B=.444; 95%CI= -.009;.896). Bisphenol concentrations were not associated with scores on attention and concentration after adjusting for confounders. CONCLUSION: In 13-15-year-old Dutch adolescents, urinary concentrations of methylparaben and ethylparaben were associated with better attention and less hyperactivity, whereas a trend toward significance was found between higher urinary propylparaben concentrations and poorer attention. Bisphenol concentrations were not associated with attention and concentration after adjusting for confounders.

Reference intervals for plasma, urinary, and salivary concentrations of free metanephrines in dogs: Relevance to the diagnosis of pheochromocytoma.

BACKGROUND: Measurement of free metanephrines is recommended for screening of pheochromocytoma (PCC) but requires appropriate reference intervals (RIs). HYPOTHESIS/OBJECTIVES: To report RIs for plasma, urinary and salivary concentrations of free metanephrines and to determine the diagnostic performance of plasma free normetanephrine (pNMN) and metanephrine (pMN) concentrations in dogs with PCC, hypercortisolism (HC), and nonadrenal illness (NAI). ANIMALS: Eighty healthy dogs, 11 PCC dogs, 25 HC dogs, 6 NAI dogs. METHODS: Plasma, urine, and saliva were collected prospectively from healthy dogs, and free metanephrine concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, medical records of dogs that had plasma free metanephrine concentrations measured by LC-MS/MS between 2018-2021 were studied retrospectively. RESULTS: The RIs for free metanephrines in plasma, urine and saliva are reported. Dogs with PCC had significantly higher pNMN than dogs with HC (P < .001) and NAI (P = .002). The PCC dogs had significantly higher pMN than HC dogs (P < .001), but not higher than NAI dogs (P = .29). Using the upper reference limit, pNMN (>3.56 nmol/L) showed high sensitivity (100%, 95% confidence interval [CI]: 72-100) and specificity (94%, 95% CI: 79-99) for diagnosis of PCC, whereas pMN (>2.49 nmol/L) showed moderate sensitivity (73%, 95% CI: 39-94) and high specificity (94%, 95% CI: 79-99). CONCLUSIONS AND CLINICAL IMPORTANCE: With establishment of these RIs, biochemical testing for PCC in dogs can be substantially improved. Measurement of pNMN is superior to pMN in dogs with PCC.

Systematic analysis of relationships between plasma branched-chain amino acid concentrations and cardiometabolic parameters: an association and Mendelian randomization study.

BACKGROUND: Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) are essential amino acids that are associated with an increased risk of cardiometabolic diseases (CMD). However, there are still only limited insights into potential direct associations between BCAAs and a wide range of CMD parameters, especially those remaining after correcting for covariates and underlying causal relationships. METHODS: To shed light on these relationships, we systematically characterized the associations between plasma BCAA concentrations and a large panel of 537 CMD parameters (including atherosclerosis-related parameters, fat distribution, plasma cytokine concentrations and cell counts, circulating concentrations of cardiovascular-related proteins and plasma metabolites) in 1400 individuals from the Dutch population cohort LifeLines DEEP and 294 overweight individuals from the 300OB cohort. After correcting for age, sex, and BMI, we assessed associations between individual BCAAs and CMD parameters. We further assessed the underlying causality using Mendelian randomization. RESULTS: A total of 838 significant associations were detected for 409 CMD parameters. BCAAs showed both common and specific associations, with the most specific associations being detected for isoleucine. Further, we found that obesity status substantially affected the strength and direction of associations for valine, which cannot be corrected for using BMI as a covariate. Subsequent univariable Mendelian randomization (UVMR), after removing BMI-associated SNPs, identified seven significant causal relationships from four CMD traits to BCAA levels, mostly for diabetes-related parameters. However, no causal effects of BCAAs on CMD parameters were supported. CONCLUSIONS: Our cross-sectional association study reports a large number of associations between BCAAs and CMD parameters. Our results highlight some specific associations for isoleucine, as well as obesity-specific effects for valine. MR-based causality analysis suggests that altered BCAA levels can be a consequence of diabetes and alteration in lipid metabolism. We found no MR evidence to support a causal role for BCAAs in CMD. These findings provide evidence to (re)evaluate the clinical importance of individual BCAAs in CMD diagnosis, prevention, and treatment.

Kynurenine metabolites predict survival in pulmonary arterial hypertension: A role for IL-6/IL-6Rα.

Activation of the kynurenine pathway (KP) has been reported in patients with pulmonary arterial hypertension (PAH) undergoing PAH therapy. We aimed to determine KP-metabolism in treatment-naïve PAH patients, investigate its prognostic values, evaluate the effect of PAH therapy on KP-metabolites and identify cytokines responsible for altered KP-metabolism. KP-metabolite levels were determined in plasma from PAH patients (median follow-up 42 months) and in rats with monocrotaline- and Sugen/hypoxia-induced PH. Blood sampling of PAH patients was performed at the time of diagnosis, six months and one year after PAH therapy. KP activation with lower tryptophan, higher kynurenine (Kyn), 3-hydroxykynurenine (3-HK), quinolinic acid (QA), kynurenic acid (KA), and anthranilic acid was observed in treatment-naïve PAH patients compared with controls. A similar KP-metabolite profile was observed in monocrotaline, but not Sugen/hypoxia-induced PAH. Human lung primary cells (microvascular endothelial cells, pulmonary artery smooth muscle cells, and fibroblasts) were exposed to different cytokines in vitro. Following exposure to interleukin-6 (IL-6)/IL-6 receptor α (IL-6Rα) complex, all cell types exhibit a similar KP-metabolite profile as observed in PAH patients. PAH therapy partially normalized this profile in survivors after one year. Increased KP-metabolites correlated with higher pulmonary vascular resistance, shorter six-minute walking distance, and worse functional class. High levels of Kyn, 3-HK, QA, and KA measured at the latest time-point were associated with worse long-term survival. KP-metabolism was activated in treatment-naïve PAH patients, likely mediated through IL-6/IL-6Rα signaling. KP-metabolites predict response to PAH therapy and survival of PAH patients.

Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC-MS/MS.

The human growth hormone GH1 (22 kDa) is a commonly measured biomarker for diagnosis and during treatment of growth disorders, but its quantification by ligand binding assays may be compromised by the occurrence of a number of isoforms. These can interfere in the assays and lead to differences in results between laboratories and potentially even in the treatment of patients. We present an LC-MS/MS method that is able to distinguish the major growth hormone isoform (GH1, 22 kDa) from other isoforms and quantify it without any interference across the clinically relevant concentration range of 0.5 to 50 ng/mL. Analysis involves purification of a 100-µL serum sample by immunocapture using an anti-GH-directed antibody, tryptic digestion, and LC-MS/MS quantification of an isoform-specific signature peptide for GH1 (22 kDa). A tryptic peptide occurring in all GH isoforms is monitored in the same 16-min analytical run as a read-out for total GH. Stable-isotope-labeled forms of these two peptides are included as internal standards. Full validation of the method according to recent guidelines, against a recombinant form of the analyte in rat plasma calibrators, demonstrated intra-assay and inter-assay imprecision below 6% across the calibration range for both signature peptides and recoveries between 94 and 102%. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for GH1 (22 kDa). Addition of up to 1000 ng/mL biotin or the presence of a 100-fold excess of GH binding protein did not affect the measurement. Equivalent method performance was found for analysis of GH in serum, EDTA, and heparin plasma. Analyte stability was demonstrated during all normal sample storage conditions. Comparison with the IDS-iSYS GH immunoassay showed a good correlation with the LC-MS/MS method for the isoform-specific signature peptide, but a significant positive bias was observed for the LC-MS/MS results of the peptide representing total GH. This seems to confirm the actual occurrence of other GH isoforms in serum. Finally, in serum from pregnant individuals, no quantifiable GH1 (22 kDa) was found, but relatively high concentrations of total GH.