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Aim: Bile acids (BAs) are implicated in the pathogenesis of several metabolic syndrome‐related diseases, including insulin resistance (IR) and type 2 diabetes (T2D). It has been reported that IR and T2D are associated with an increased ratio of 12α/non‐12α‐hydroxylated BAs in the circulating BA pool. It is, however, unknown whether the improvement of insulin sensitivity inversely affects BA composition in humans. Therefore, we assessed whether lifestyle‐induced weight loss induces changes in BA metabolism in people with obesity, with or without T2D, and if these changes are associated with metabolic parameters. Materials and Methods: Individual BAs and C4 were quantified by ultra‐high‐performance liquid chromatography‐tandem mass spectrometry in plasma samples collected from two cohorts of people with obesity (OB) and with T2D and obesity (T2D), before and after a lifestyle intervention. Results: Lifestyle‐induced weight loss improved glycaemic control in both cohorts, with plasma BA concentrations not affected by the lifestyle interventions. The ratio of 12α/non‐12α‐hydroxylated BAs remained unchanged in OB (p =.178) and even slightly increased upon intervention in T2D (p =.0147). Plasma C4 levels were unaffected in OB participants (p =.20) but significantly reduced in T2D after intervention (p =.0003). There were no significant correlations between the ratio of 12α/non‐12α‐hydroxylated BAs and glucose, insulin, or homeostatic model assessment‐IR, nor in plasma triglycerides, low‐density lipoprotein cholesterol, lipoprotein (a) in the T2D cohort. Conclusions: Lifestyle‐induced weight loss did improve glycaemic control but did not affect BA concentrations. Improvements in insulin sensitivity were not associated with changes in BA parameters in people with obesity, with or without T2D. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Trimethylamine N‐oxide (TMAO) is a circulating microbiome‐derived metabolite implicated in the development of atherosclerosis and cardiovascular disease (CVD). We investigated whether plasma levels of TMAO, its precursors (betaine, carnitine, deoxycarnitine, choline), and TMAO‐to‐precursor ratios are associated with clinical outcomes, including CVD and mortality. This was followed by an in‐depth analysis of their genetic, gut microbial, and dietary determinants. The analyses were conducted in five Dutch prospective cohort studies including 7834 individuals. To further investigate association results, Mendelian Randomization (MR) was also explored. We found only plasma choline levels (hazard ratio [HR] 1.17, [95% CI 1.07; 1.28]) and not TMAO to be associated with CVD risk. Our association analyses uncovered 10 genome‐wide significant loci, including novel genomic regions for betaine (6p21.1, 6q25.3), choline (2q34, 5q31.1), and deoxycarnitine (10q21.2, 11p14.2) comprising several metabolic gene associations, for example, CPS1 or PEMT. Furthermore, our analyses uncovered 68 gut microbiota associations, mainly related to TMAO‐to‐precursors ratios and the Ruminococcaceae family, and 16 associations of food groups and metabolites including fish‐TMAO, meat‐carnitine, and plant‐based food‐betaine associations. No significant association was identified by the MR approach. Our analyses provide novel insights into the TMAO pathway, its determinants, and pathophysiological impact on the general population. Highlights: Exploration of microbiome‐related metabolites (trimethylamine N‐oxide [TMAO], choline, betaine, l‐carnitine, and deoxycarnitine) in 7834 participants from five population cohorts.Cardiovascular risk was associated with elevated choline concentrations, but not with TMAO concentrations.Characterization of the genetic architecture behind metabolite concentration variability.Identification of gut microbial taxonomic abundance associated with metabolite's plasma concentration levels.Fish intake is the major dietary driver of TMAO concentrations, and betaine is related to grains and vegetable intake. [ABSTRACT FROM AUTHOR]

In birds, exposure to maternal (yolk) testosterone affects a div ersity of offspring post-hatching traits, which eventually affect offspring competiti veness. 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 chr omatography 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 limita tion. 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 t akes place here. We also found no sex-specific metabolism, explaining why maternal t estosterone does not affect sexual differentiation. Our findings showed that maternal t estosterone is quickly converted by the embryo, with several but not all metabolites reaching the embryo providing evidence for both hypotheses. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Patients with adrenal insufficiency are treated with oral hydrocortisone (HC) to compensate for the loss of endogenous cortisol production. Intrinsic imperfections of cortisol replacement strategies in mimicking normal cortisol secretion are the underlying cause of the increased morbidity and mortality of patients suffering from secondary adrenal insufficiency (SAI). To improve oral hydrocortisone substitution therapy, a better understanding of its pharmacokinetics (PK) is necessary. The previous PK model did not include protein binding. It is known that protein binding can impact hydrocortisone pharmacokinetics. The aim of this study is to describe HC pharmacokinetics including the protein-binding state using Edsim++ (Mediware, Prague) pharmacokinetic modeling software, paving the way for an in-silico tool suitable for drug delivery design. Methods: A total of 46 patients with SAI participated in a randomized double-blind crossover study Patients randomly received a low dose of HC (0.2–0.3 mg/kg body weight/day) for 10 weeks, followed by a high dose (0.4–0.6 mg/kg body weight/day) for another 10 weeks, or vice versa. Plasma samples were obtained and analyzed for free and total hydrocortisone. Single compartment population pharmacokinetic analysis was performed using an extended Werumeus-Buning model built in Edsim++. This model includes a mathematical approach for estimating free cortisol by Nguyen et al., taking the protein binding of HC to albumin and hydrocortisone-binding globulin (CBG, transcortin) into consideration, as well as different states of CBG which affect binding kinetics to HC. The goodness of fit for observed versus predicted values was calculated. Results and conclusions: Nguyen's formula for free cortisol estimation was successfully implemented in a pharmacokinetic model. The model shows high Spearman's correlation for observed versus predicted hydrocortisone concentrations. Significantly higher correlations (Spearman's r, 0.901 vs. 0.836) between total and free hydrocortisone AUC24 (area-under the curve over 24 h) are found when comparing new and old models. This new model was used to simulate the plasma concentration–time behavior of a more suitable hydrocortisone formulation. [ABSTRACT FROM AUTHOR]

Objective: To compare cortisol pharmacokinetics and pharmacodynamics mapped through several glucocorticoid sensitive pathways in patients on hydrocortisone substitution with or without an adrenal crisis. Design: A post-hoc analysis of a previously conducted randomized controlled trial in patients with secondary adrenal insufficiency examining the effects of 2 weight-adjusted hydrocortisone doses. Methods: Comparisons were primarily made on a hydrocortisone dose of 0.2-0.3 mg/kg/day for plasma cortisol and cortisone, 24-hour urinary steroid profile, the glucocorticoid sensitive tryptophan-kynurenine pathway, the renin-angiotensin-aldosterone system and aspects of quality of life. Variables of interest were also analyzed on the hydrocortisone dose of 0.4-0.6 mg/kg/day. Results: Out of 52 patients, 9 (17%) experienced at least one adrenal crisis (AC+ group) and 43 did not develop an adrenal crisis (AC- group) during an observation period of 10 years. 24-hour urinary excretion of cortisol and cortisone were lower in the AC+ group (0.05 [IQR 0.03; 0.05] vs. 0.09 [0.05; 0.12] µmol/24h, P=0.01and 0.13 [0.10; 0.23] vs. 0.24 [0.19; 0.38] µmol/24h, P=0.04, respectively). No differences in pharmacokinetics of cortisol were observed. Kynurenine concentrations were higher in the AC+ group (2.64 [2.43; 3.28] vs. 2.23 [1.82; 2.38] µmol/L, P=0.03) as was general fatigue (Z-scores 1.02 [-0.11; 1.42] vs. -0.16 [- 0.80; 0.28], P=0.04). On the higher hydrocortisone dose urinary excretion of cortisol and cortisone was still significantly lower between the AC- and AC + group. The differences in glucocorticoid sensitive variables disappeared. Conclusion: Patients susceptible to an adrenal crisis demonstrated differences in cortisol and cortisone excretion as well as in pharmacodynamics when compared to patients who did not experience an adrenal crisis, suggesting a biological predisposition in certain patients for the development of an adrenal crisis. [ABSTRACT FROM AUTHOR]

Background: Phototherapy is used on the majority of preterm infants with unconjugated hyperbilirubinaemia. The use of fluorescent tube phototherapy is known to induce oxidative DNA damage in infants and has largely been replaced by blue light-emitting diode phototherapy (BLP). To date, it is unknown whether BLP also induces oxidative DNA damage in preterm infants.Objective: To determine whether BLP in preterm infants induces oxidative DNA damage as indicated by 8-hydroxy-2'deoxyguanosine (8-OHdG).Design: Observational cohort study.Methods: Urine samples (n=481) were collected in a cohort of 40 preterm infants (24-32 weeks' gestational age) during the first week after birth. Urine was analysed for the oxidative marker of DNA damage 8-OHdG and for creatinine, and the 8-OHdG/creatinine ratio was calculated. Durations of phototherapy and levels of irradiance were monitored as well as total serum bilirubin concentrations.Results: BLP did not alter urinary 8-OHdG/creatinine ratios (B=0.2, 95% CI -6.2 to 6.6) at either low (10-30 µW/cm2/nm) or high (>30 µW/cm2/nm) irradiance: (B=2.3, 95% CI -5.7 to 10.2 and B=-3.0, 95% CI -11.7 to 5.6, respectively). Also, the 8-OHdG/creatinine ratios were independent on phototherapy duration (B=-0.1, 95% CI -0.3 to 0.1).Conclusions: BLP at irradiances up to 35 µW/cm2/nm given to preterm infants ≤32 weeks' gestation does not affect 8-OHdG, an oxidative marker of DNA damage. [ABSTRACT FROM AUTHOR]