Martijn van Faassen

Background: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a rare disease affecting the ABCB11 gene, encoding the bile salt export pump (BSEP). BSEP dysfunction impairs bile acid (BA) secretion, causing hepatic damage and leading to pruritus, cholestasis, hepatomegaly, and often fibrosis and end-stage hepatic disease. Treatments include ileal BA transporter inhibitors, surgical bile diversion, or ultimately, liver transplantation. Our aim was to develop a PFIC2 gene therapy approach based on the restoration of BSEP hepatocyte expression. Methods and Results: We designed several expression cassettes containing the human ABCB11 gene downstream of either a liver-specific constitutive promoter or a BA-inducible promoter. After in vitro screening, the AAV vectors with the best expression cassette for each promoter were tested in a PFIC2 mouse model. The AAV vector containing the constitutive promoter, named VTX-802, showed higher BSEP expression, resulting in better restoration of BA secretion 3 and 7 weeks post-treatment. We then performed a dose-range-finding study of VTX-802 in 5-week-old female PFIC2 mice in which the therapeutic efficacy was monitored until 5 months of age. Treated mice showed a sustained dose-dependent improvement in serum transaminase levels. These mice also exhibited significant, but partial, correction of hepatomegaly, and increased BA levels in bile and small intestine, indicating partial restoration of normal BA secretion. Conclusions: VTX-802 restores hepatic BSEP expression and partially corrects disease phenotype in PFIC2 mice. To our knowledge, VTX-802 is the first gene therapy approach that could potentially benefit PFIC2 patients.

Context: The hypothalamic–pituitary–adrenal (HPA) axis is the key homeostatic system regulating the response to surgical stress. Imbalances in HPA axis hormones increase morbidity and mortality in children after cardiac surgery. Despite this, the physiology of the HPA axis in children undergoing cardiac surgery is poorly understood, leading to controversies in clinical practice. Objective: To characterize dynamic HPA axis responses in children undergoing cardiac surgery and to determine age- and procedure-related differences in cortisol and cortisone physiology. Methods: We recruited children (0-18 years) undergoing cardiac surgery with cardiopulmonary bypass or cardiac catheterization. Tissue-free cortisol and cortisone were sampled every 20 minutes for up to 24 hours via microdialysis, alongside serum adrenocorticotropic hormone (ACTH), cortisol, cortisol-binding globulin (CBG), and inflammatory markers. We developed dynamic markers to quantify age- and procedure-dependent differences in hormonal responses and built a mathematical model to explain them. Results: Neonates undergoing surgery showed higher free cortisol and cortisone AUC and peak concentrations than catheterization patients. Neonates had higher peaks of cortisol and cortisone than older children undergoing surgery. The much higher tissue cortisone levels observed in neonates can be explained by enzymatic interconversion between cortisol and cortisone, likely due to persistent fetal high 11-βHSD2 activity and reduced 11-βHSD1 activity. Low postoperative blood cortisol and CBG values in neonates resulted in high free cortisol peaks in interstitial fluid during and after surgery. Conclusion: Neonates differ physiologically, with higher free cortisol levels that more readily diffuse into interstitial tissues, with implications for perioperative management.

Background: Accurate interpretation of insulin-like growth factor-1 (IGF1) requires both analytically robust measurement and appropriate reference data. Most available IGF1 normative datasets are based on ligand-binding assays (LBA), limiting their applicability to liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods that provide higher analytical specificity. Objective: To evaluate the clinical performance of an in-house developed LC-MS/MS IGF1 assay in combination with a method-matched normative dataset derived from the Dutch Lifelines cohort, and to compare it with a commonly used LBA. Methods: Paired IGF1 results (n = 2057) were obtained using LC-MS/MS and the IDS iSYS LBA. Standard deviation scores (SDS) were calculated using assay-specific normative datasets. Agreement was assessed by regression, Bland-Altman, and weighted Cohen's κ analyses. Clinical validity was evaluated by comparing extreme SDS values with diagnostic categories adjudicated by endocrinologists. Results: The LC-MS/MS method showed a proportional bias of −27% versus IDS iSYS and yielded symmetrically distributed SDS around zero, whereas IDS iSYS results were positively skewed. Categorical agreement was fair (κ = 0.39). LC-MS/MS-derived SDS corresponded more closely with clinical diagnoses across adult and pediatric groups, including acromegaly, growth hormone (GH) deficiency, and GH replacement therapy. Conclusions: Our LC-MS/MS IGF1 assay integrated with its own population-based normative dataset demonstrated superior alignment with clinical assessment compared with LBA results. In our setting, this method provides a clinically reliable benchmark for IGF1 interpretation and may serve as a reference for IGF1 harmonization and broader adoption of the Lifelines normative dataset in routine endocrinology.

Objectives: Primary aldosteronism (PA) is a common cause of secondary hypertension. To address the specificity limits of immunoassays, we developed and validated an LC-MS/MS method for urinary aldosterone and tetrahydroaldosterone and established method-matched reference intervals for excretion in 24 h urine. Methods: Urinary aldosterone, tetrahydroaldosterone and their glucuronidated metabolites were extracted in the presence of internal standards using offline solid-phase extraction (SPE), followed by enzymatic hydrolysis to release the glucuronidated fraction. Subsequently, aldosterone and tetrahydroaldosterone were analyzed by online SPE in combination with LC-MS/MS. Reference intervals were established based on 24 h urine samples from 265 individuals participating in the Lifelines Cohort study. Results: Intra- and inter-assay imprecision ranged from 2.0–12.3 % for aldosterone, and 1.3–6.3 % for tetrahydroaldosterone. The lower limits of quantification were 0.44 nmol/L and 0.10 nmol/L, respectively. Recoveries ranged from 97–106 %, calibration was linear, with correlation coefficients greater than 0.999, and no carry-over was observed. Total aldosterone concentrations measured by LC-MS/MS were consistently higher than those obtained by radioimmunoassay. In the reference population, 24 h urinary excretion ranged from 5.4–76.7 nmol/24 h for aldosterone and 21.4–269.9 nmol/24 h for tetrahydroaldosterone. Conclusions: This validated LC-MS/MS assay, together with a method-matched normative dataset, enables standardized urinary aldosterone profiling and defines reference intervals that will help improve the interpretability of results in the biochemical diagnosis of PA.

Mitochondria are essential organelles for cellular energy production, playing a central role in driving metabolic processes and supporting critical intracellular functions. Neurometabolic disorders encompass a wide variety of conditions characterized by mitochondrial dysfunction. Owing to their bacterial ancestry, mitochondria possess an independent genome consisting of a circular DNA molecule (mtDNA), which has been reported to be subject to methylation. However, the technical challenges in the detection of mtDNA methylation have led to debates on its existence. One of the concerns is that the compactness of mtDNA can lead to suboptimal bisulfite conversion, thereby causing mtDNA methylation overestimation. To address this, liquid chromatography tandem mass spectrometry (LC-MS/MS) offers a bisulfite-independent readout; however, this method requires mtDNA samples devoid of nuclear DNA (nDNA) contamination. To diminish nDNA contamination, we isolated mtDNA from the TRIzol RNA phase. Importantly, pyrosequencing showed no significant difference in the methylation levels of mtDNA isolated from the TRIzol RNA phase compared to those from the TRIzol DNA phase, or isolated via total genomic DNA (gDNA). Across different human cell lines, LC-MS/MS detected significantly lower global methylation levels for DNA isolated from the TRIzol RNA phase than those from the TRIzol DNA or gDNA isolation. Moreover, using mtDNA isolated from the TRIzol RNA phase, LC-MS/MS validated the enhanced mtDNA methylation in HepG2 transgenic cell lines expressing mitochondrial-targeted DNA methyltransferases (means of 2.89% and 2.03% for MCviPI and MSssI transgenic cell lines, respectively), compared to two negative control cell lines (1.36 and 1.39%). When applying it to clinically relevant material, LC-MS/MS demonstrated a significantly lower global methylation level for platelet DNA isolated from the TRIzol RNA phase (mean of 1.98%) compared to gDNA isolations (mean of 4.32%). Similar findings were confirmed in mouse brain tissue, in which a significantly lower methylation level was detected in DNA isolated from the TRIzol RNA phase (1.79%) compared to that from gDNA isolation (5.12%). In conclusion, isolating mtDNA from the TRIzol RNA phase holds significant potential in future studies, particularly for the quantification of mtDNA global methylation by LC-MS/MS, a technique that is independent of bisulfite conversion and bioinformatic analysis.

Background: Tryptophan and its kynurenine pathway (KP) metabolites play key roles in modulating the immune system and vasculature, and exhibit both pro- and antioxidant properties, making them crucial for a healthy pregnancy and fetal development. Disruptions in the KP may impact both prenatal and postnatal health, however, data on fetal KP metabolite concentrations and their alterations in pregnancy-related disorders remain scarce. This study aims to investigate the association between pregnancy complications and KP metabolite concentrations in umbilical cord blood. Methods: Pregnancies complicated by preeclampsia (n = 40), fetal growth restriction (FGR, n = 33), pregestational diabetes mellitus (DM, n = 42), gestational diabetes mellitus (GDM, n = 61), and amniotic infection syndrome (AIS, n = 47) were included, along with 410 controls matched in a 1:2 ratio using Mahalanobis nearest-neighbor matching from a prospective birth cohort study. Tryptophan, kynurenine, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, kynurenic acid, xanthurenic acid, quinolinic acid, picolinic acid, and nicotinic acid were measured in umbilical cord blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differences in metabolite concentrations were analyzed using unpaired t-tests and linear regression models to control for potential confounders. Results: Tryptophan concentrations were decreased in cases of preeclampsia and DM. We identified elevated levels of 3-hydroxykynurenine in preeclampsia, kynurenine in GDM, and nicotinic acid in both FGR and DM. Quinolinic acid levels were also higher in preeclampsia and GDM, although this was not significant after adjusting for confounding variables. We observed no changes in KP metabolites in AIS. Conclusion: This study identified distinct alterations in umbilical cord blood KP metabolite concentrations in pregnancies with preeclampsia, FGR, DM, and GDM, but not AIS. This suggests differential regulation and activation of the KP depending on the pregnancy disorder. Such changes may influence maternal and infant health and could play a role in fetal programming, with potential long-term effects on child development and health.

Background: Given the lack of effective medical treatment for pheochromocytomas (PCCs), a reliable in vitro model is needed to explore new therapies. Organoids are three-dimensional self-renewing structures that exhibit key features of their tissue of origin, providing valuable platforms for disease modeling and drug screening. Aim: This study aimed to establish and characterize organoid cultures of canine normal adrenal medullas and PCCs. Methods: Normal adrenal medullas from healthy dogs and tumor tissue from client-owned dogs with PCC were used to develop organoids. Primary cell suspensions were cultured in a 3D matrix, and organoids were established under optimized conditions. Organoids were characterized using histology, immunohistochemistry, immunofluorescence, qPCR, and metanephrine analysis by LC-MS/MS. Results: Five adrenomedullary organoid lines were successfully established, demonstrating sustained growth. Organoid cultures were also derived from nine PCCs, although expansion was limited after passages 1-2. Both adrenomedullary and PCC organoids expressed differentiation markers (chromogranin A, synaptophysin, phenylethanolamine N-methyltransferase) and stem/progenitor markers (nestin, SOX10). Organoids retained key functional traits, as indicated by metanephrine levels in culture supernatants, which initially mirrored primary tumor patterns. A decline in both differentiation marker expression and metanephrine levels was observed over time, possibly due to organoid dedifferentiation or selective loss of differentiated chromaffin cells. Conclusions: This study demonstrates the establishment of the first adrenomedullary and PCC organoid lines. While further optimization is needed, these organoids offer valuable potential as an in vitro model to investigate PCC pathophysiology and explore novel treatment strategies for this therapeutically challenging tumor.

Cognitive and psychiatric problems are common in cancer patients, but literature on patients with neuroendocrine tumors (NET) is scarce. In a subset of these patients, the tumor produces serotonin, causing physical symptoms known as carcinoid syndrome. This peripheral overproduction of serotonin may cause central depletion of its precursor tryptophan, potentially resulting in cognitive and psychiatric problems. Therefore, we investigated cognitive and psychiatric function in patients with a serotonin overproduction and the association with this serotonin overproduction. Eighty-one patients with a serotonin-producing metastatic ileal NET underwent standardized neuropsychological and psychiatric assessment. Blood and urine samples were collected to determine concentrations of serotonin, its precursor tryptophan, and metabolite (5-HIAA). Multivariate normative comparison was applied to determine the prevalence of cognitive impairment. Separate linear regressions of serotonin, tryptophan, and 5-HIAA concentrations on cognitive function, depressive symptoms, and anxiety symptoms were performed, corrected for age, sex, education, and/or duration of illness. We found an 11% prevalence of cognitive impairment and a 20% prevalence of psychiatric disorders. Cognitive function was not related to measures of peripheral serotonin production. Unexpectedly, depressive symptoms were significantly associated with lower serum serotonin concentrations and elevated serum tryptophan concentrations. Cognitive symptoms of anxiety were also associated with elevated tryptophan concentrations. Concluding, cognitive or psychiatric problems occur in a minority of patients with NET and cannot be explained by tryptophan depletion following tumor-related serotonin production.

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 (r_s = −0.212, P = .005; r_s = −0.182, P = .016; and r_s = −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) (r_s = 0.146, P = .01; r_s = 0.151, P = .009; and r_s = 0.180, P = .002, respectively). Concentrations of UCB metanephrine and 3-methoxytyramine were negatively correlated with the lowest measured BG concentration (r_s = −0.149, P = .01; and r_s = −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 β-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.