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.

SHBG gene polymorphisms and their influence on serum SHBG, total and free testosterone concentrations in men.

CONTEXT: Genetic variation in sex hormone-binding globulin (SHBG) structure may affect estimates of sex steroid exposure by altering the affinity of the protein for its ligand. Consequently, free hormone calculations assuming constant binding affinity may, for certain genetic variations, lead to incorrect diagnoses if genetic variation is not taken into consideration. OBJECTIVE: To investigate the effects of genetic variation in SHBG on calculated and measured serum free testosterone (T) in men. DESIGN, SETTING AND PARTICIPANTS: Population-based sibling-pair study in 999 healthy men aged 25 to 45 (mean: 34.5) years. MAIN OUTCOME MEASURES: Genotyping using microarray (Illumina®) for SNPs suggested to affect binding affinity and/or concentration of SHBG or T. SHBG concentrations were measured using immunoassay and in a subset (n = 32) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total T was measured using LC-MS/MS. Free T was calculated and in a subset (n = 314) measured directly using LC-MS/MS after equilibrium dialysis. RESULTS: Allelic frequencies of analyzed SNPs ranged from 0.5% to 58.2%. Compared to wild-type, SHBG concentrations were lower in rs6258 heterozygotes (-24.7%; p < 0.05) and higher in rs6259 heterozygotes, rs727428 homozygotes, and carriers of rs1799941 (+10.8 to 23.1%; all p < 0.05). Total T was higher in rs727428 homozygotes and carriers of rs5934505, rs1799941and rs6259 (+3.9 to 21.4%; all p < 0.05). No clear effects on measured free T were found, except for a trend towards higher values in rs6259 homozygotes, significant for calculated free T (+18.7%; p < 0.05) in the larger global study population. CONCLUSION: In these men, analyzed SNPs were relatively prevalent and affected serum concentrations of total T and SHBG but not calculated or measured free T except for a higher trend in rs6259 homozygotes.

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.

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.

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.

Hereditary Alpha Tryptasemia: Validation of a Single-Well Multiplex Digital Droplet PCR Assay in a Cohort of Symptomatic Patients.

BACKGROUND: Hereditary alpha tryptasemia (HαT) has significant prevalence and potential morbidity in the general population. However, it remains largely undiagnosed in routine clinical diagnostics due to low availability of efficient assessment methods. To address this issue, we developed a reliable and efficient single-well multiplex digital droplet PCR assay. METHODS: The assay was based on the reconstruction of the TPSAB1 gene through quantification of the ratio of α- and ß-tryptase copy number variants (CNV) in a single-well measurement. We performed analytical validation by determining CNV measurement clustering around the expected copy numbers in 281 cases and determined the diagnostic accuracy of basal serum tryptase (BST) to predict HαT and HαT subtypes in 141 symptomatic patients. RESULTS: The assay determined α- and ß-tryptase CNVs with an overall accuracy, expressed as a 99% prediction interval, of 0.03 ± 0.27 copy numbers. The optimal BST cutoff level to predict HαT in symptomatic patients, who had no other explanation for relatively high tryptase levels (i.e., no diagnosis of systemic mastocytosis, myeloid neoplasm, or end-stage renal failure), was 9.2 ng/mL (sensitivity: 98.1%; specificity: 96.6%). HαT showed a linear gene-dose effect, with an average gene-dose increase of 7.5 ng/mL per extra α-tryptase gene. CONCLUSION: Our single-well multiplex digital droplet PCR assay accurately determined HαT and could be implemented as a state-of-the-art routine diagnostic test. The assay demonstrated a strong correlation with BST and the optimal threshold for identifying HαT in symptomatic patients with unexplained high tryptase concentrations was at a BST level of 9.2 ng/mL.

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.

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.

Quantitative bioanalysis of proteins by digestion and LC-MS/MS: the use of multiple signature peptides.

The use of multiple signature peptides for the quantification of proteins by digestion and LC-MS/MS is reviewed and evaluated here. A distinction is made based on the purpose of the use of multiple peptides: confirmation of the protein concentration, discrimination between different protein forms or species and in vivo biotransformation. Most reports that describe methods with at least two peptides use these for confirmation, but it is not always mentioned how the peptides are used and how possible differences in concentration between the peptides are handled. Differences in concentration are often reported in the case of monitoring different protein forms or in vivo biotransformation, and this offers insight into the biological fate of the protein.

Sleep quality, fatigue, societal participation and health-related quality of life in kidney transplant recipients: a cross-sectional and longitudinal cohort study.

BACKGROUND: Fatigue and impaired health-related quality of life (HRQoL) are common among kidney transplant recipients (KTR). We hypothesized that both may partially be attributable to poor sleep. METHODS: Cross-sectional and longitudinal data of KTR enrolled in the TransplantLines Biobank and Cohort Study were used. Sleep quality was assessed using the Pittsburgh Sleep Quality Index questionnaire. Individual strength (i.e. a composite of fatigue, concentration, motivation and physical activity), societal participation and HRQoL were assessed using validated questionnaires. RESULTS: We included 872 KTR (39% female, age 56 ± 13 years) and 335 healthy controls. In total, 33% of male KTR and 49% of female KTR reported poor sleep quality, which was higher compared with male and female healthy controls (19% and 28%, respectively, P < .001 for both). In logistic regression analyses, female sex, anxiety, active smoking, low protein intake, physically inactive lifestyle, low plasma magnesium concentration, using calcineurin inhibitors, not using mTOR inhibitors and using benzodiazepine agonists were associated with poor sleep quality. In adjusted linear regression analyses, poor sleep was strongly and independently associated with lower individual strength [standardized ß (st.ß) = 0.59, 95% confidence interval (CI) 0.45 to 0.74, P < .001], poorer societal participation (frequency: st.ß = -0.17, 95% CI -0.32 to -0.01, P = .04; restrictions: st.ß = -0.36, 95% CI -0.51 to -0.21, P < .001; satisfaction: st.ß = -0.44, 95% CI -0.59 to -0.28, P < .001) and lower HRQoL (physical: st.ß = -0.53, 95% CI -0.68 to -0.38, P < .001; mental: st.ß = -0.64, 95% CI -0.78 to -0.50, P < .001). The associations with poorer societal participation and lower HRQoL were strongly mediated by individual strength (P < .001 for all), yet the suggested direct effects of poor sleep quality on HRQoL remained significant (Pphysical = .03, Pmental = .002). Longitudinal data of 292 KTR showed that sleep quality improves after kidney transplantation in males (P < .001), but not in females (P = .9). CONCLUSIONS: Poor sleep quality is common among KTR, and may be a potential target to improve fatigue, societal participation and HRQoL among KTR.

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.

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.

Circulating FGF21 Concentration, Fasting Plasma Glucose, and the Risk of Type 2 Diabetes: Results From the PREVEND Study.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) is a peptide hormone synthesized by several organs and regulates, among others, energy homeostasis. In obesity, insulin resistance and type 2 diabetes (T2D), higher circulating FGF21 concentrations have been found. Temporal analyses in murine studies demonstrate that FGF21 increases before insulin resistance occurs. The current study aims to investigate in time-to-event analyses whether FGF21 may be an early biomarker in the development of T2D. RESEARCH DESIGN AND METHODS: Circulating FGF21 was measured using an immunoassay of the Mesoscale U-PLEX assay platform. The study outcome was incident T2D. Associations of circulating FGF21 concentration with T2D were quantified using Cox proportional hazards models with adjustments for potential confounders. RESULTS: We included 5244 participants aged 52 ± 12 years, of whom 50% were male. Median [interquartile range] circulating FGF21 concentration was 860 [525-1329] pg/mL. During 7.3 [6.1-7.7] years of follow-up, 299 (5.7%) participants developed T2D. In fully adjusted analyses, higher circulating FGF21 concentration was associated with an increased risk of incident T2D (hazard ratio per doubling: 1.26 [95% CI, 1.06-1.51]; P = 0.008), with effect modification by fasting plasma glucose, consistent with strengthening of the association at lower fasting glucose (interaction coefficient: -0.12; P = 0.022). CONCLUSION: Higher circulating FGF21 concentrations are independently associated with an increased risk of incident T2D in participants with a low fasting plasma glucose, making circulating FGF21 concentration a potential early biomarker for type 2 diabetes.

Dietary lithium intake, graft failure and mortality in kidney transplant recipients.

BACKGROUND: Long-term high-dose lithium therapy in bipolar disorder is known to adversely affect kidney function. However, recent animal studies have revealed that low amounts of lithium are beneficial for the kidney when it is damaged by exposure to nephrotoxic compounds, inflammation or oxidative stress. This study aimed to investigate whether urinary lithium excretion, reflecting dietary lithium intake, is associated with adverse long-term kidney graft outcomes and patient survival. METHODS: Urinary lithium concentration was measured using inductively coupled plasma mass spectrometry in 642 stable kidney transplant recipients (KTRs). Graft failure was defined as the start of dialysis or retransplantation and kidney function decline was defined as a doubling of serum creatinine. RESULTS: The median urinary lithium excretion was 3.03 µmol/24 h [interquartile range (IQR) 2.31-4.01]. Urinary lithium excretion was associated with energy, plant protein and water intake. During a median follow-up of 5.3 years (IQR 4.5-6.0), 79 (12%) KTRs developed graft failure and 127 (20%) KTRs developed kidney function decline. Higher urinary lithium excretion was associated with a lower risk of graft failure {hazard ratio [HR] per doubling 0.54 [95% confidence interval (CI) 0.38-0.79]} and kidney function decline [HR per doubling 0.73 (95% CI 0.54-0.99)]. These associations remained independent of adjustment for potential confounders and in sensitivity analyses. There was a significant effect modification with the use of proliferation inhibitors (P = .05) and baseline estimated glomerular filtration rate (eGFR; P < .001), with higher urinary lithium excretion being more protective in KTRs not using proliferation inhibitors and in KTRs with lower baseline eGFR. Furthermore, higher urinary lithium excretion was associated with a reduced risk of all-cause mortality [HR 0.64 (95% CI 0.49-0.83); P = .001]. CONCLUSION: Dietary lithium intake may be a potentially modifiable, yet rather overlooked, risk factor for adverse long-term kidney graft outcomes and patient survival. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02811835.

Clinical irrelevance of lower titer thyroglobulin autoantibodies in patients with differentiated thyroid carcinoma.

Objective: Thyroglobulin (Tg) is an established tumor marker for differentiated thyroid carcinoma (DTC) patients. However, Tg immunoassays can be subject to Tg autoantibody (TgAb) interference resulting in incorrect Tg values. Therefore, Tg measurement with liquid chromatography-tandem mass spectrometry (LC-MS/MS) could be promising in patients with TgAbs. In this study, we compared Tg IRMA and Tg-LC-MS/MS analytically in the presence of TgAbs. Furthermore, we compared the clinical interpretation of results obtained by both Tg assays in DTC patients with lower TgAbs titers (<10 U/mL) during 131I ablation therapy. Methods: Totally 118 DTC patients diagnosed between 2006 and 2014 in a University Medical Center were followed with the Tg-IRMA (Thermo Fischer Scientific) and ARCHITECT anti-Tg (Abbott Laboratories) assays. We re-analyzed their samples with a sensitive Tg-LC-MS/MS method (Labcorp, limit of quantification of 0.02 ng/mL). Passing-Bablok regression analysis was performed on samples obtained during 131I ablation therapy and follow-up. Results: In 304 samples with lower TgAb titers, a good analytical agreement was found between both Tg assays (slope of 1.09 (95% CI: 1.05-1.16)). Fifty-five samples with potentially interfering TgAbs showed higher Tg-LC-MS/MS values than Tg-IRMA (slope of 1.45 (95% CI: 1.12->>100)). In patients(n = 91) with lower TgAb titers at the time of 131I ablation therapy, the Tg assays showed a clinical concordance of 91.2, 87.9, and 98.9%, respectively, using a Tg cut-off value of 1.0, 2.0, and 5.0 ng/mL. Conclusions: In DTC patients with lower titer TgAbs, Tg-IRMA is still a reliable and useful tumor marker. In DTC patients with potentially interfering TgAbs, Tg-IRMA values decreased due to TgAb interference.